Skp2B Overexpression Alters a Prohibitin-p53 Axis and the Transcription of PAPP-A, the Protease of Insulin-Like Growth Factor Binding Protein 4

We previously reported that the degradation of prohibitin by the SCF(Skp2B) ubiquitin ligase results in a defect in the activity of p53. We also reported that MMTV-Skp2B transgenic mice develop mammary gland tumors that are characterized by an increased proteolytic cleavage of the insulin-like growth factor binding protein 4 (IGFBP-4), an inhibitor of IGF signaling. However, whether a link exists between a defect in p53 activity and proteolysis of IGFBP-4 was not established.We analyzed the levels of pregnancy-associated plasma protein A (PAPP-A), the protease of IGFBP-4, in MMTV-Skp2B transgenic mice and found that PAPP-A levels are elevated. Further, we found a p53 binding site in intron 1 of the PAPP-A gene and that both wild type and mutant p53 bind to this site. However, binding of wild type p53 results in the transcriptional repression of PAPP-A, while binding of mutant p53 results in the transcriptional activation of PAPP-A. Since MMTV-Skp2B mice express wild type p53 and yet show elevated levels of PAPP-A, at first, these observations appeared contradictory. However, further analysis revealed that the defect in p53 activity in Skp2B overexpressing cells does not only abolish the activity of wild type of p53 but actually mimics that of mutant p53. Our results suggest that in absence of prohibitin, the half-life of p53 is increased and like mutant p53, the conformation of p53 is denatured.These observations revealed a novel function of prohibitin as a chaperone of p53. Further, they suggest that binding of denatured p53 in intron 1 causes an enhancer effect and increases the transcription of PAPP-A. Therefore, these findings indicate that the defect in p53 function and the increased proteolysis of IGFBP-4, we had observed, represent two components of the same pathway, which contributes to the oncogenic function of Skp2B

Haptoglobin phenotype is not a predictor of recurrence free survival in high-risk primary breast cancer patients

Contains fulltext : 70104tjan-heijnen.pdf (publisher's version ) (Open Access)BACKGROUND: Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective follow-up study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence free survival. METHODS: Two sample sets of high-risk primary breast cancer patients participating in a randomised national trial investigating the effectiveness of high-dose chemotherapy were analysed. Sera in set I (n = 63) were analysed by surface enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF MS) for biomarker finding. Initial results were validated by analysis of sample set II (n = 371), using one-dimensional gel-electrophoresis. RESULTS: In sample set I, the expression of a peak at mass-to-charge ratio 9198 (relative intensity 20), identified as haptoglobin (Hp) alpha-1 chain, was strongly associated with recurrence free survival (global Log-rank test; p = 0.0014). Haptoglobin is present in three distinct phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2), of which only individuals with phenotype Hp 1-1 or Hp 2-1 express the haptoglobin alpha-1 chain. As the expression of the haptoglobin alpha-1 chain, determined by SELDI-TOF MS, corresponds to the phenotype, initial results were validated by haptoglobin phenotyping of the independent sample set II by native one-dimensional gel-electrophoresis. With the Hp 1-1 phenotype as the reference category, the univariate hazard ratio for recurrence was 0.87 (95% CI: 0.56 - 1.34, p = 0.5221) and 1.03 (95% CI: 0.65 - 1.64, p = 0.8966) for the Hp 2-1 and Hp 2-2 phenotypes, respectively, in sample set II. CONCLUSION: In contrast to our initial results, the haptoglobin phenotype was not identified as a predictor of recurrence free survival in high-risk primary breast cancer in our validation set. Our initial observation in the discovery set was probably the result of a type I error (i.e. false positive). This study illustrates the importance of validation in obtaining the true clinical applicability of a potential biomarker

Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

BACKGROUND:Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. METHODOLOGY/PRINCIPAL FINDING:Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c-VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. SIGNIFICANCE:Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis

Adiponectin and cancer: a systematic review

Recent studies have demonstrated that obesity is a significant risk factor for the development of several malignancies, but the mechanisms underlying this relationship remain to be fully elucidated. Adiponectin, an adipocyte secreted endogenous insulin sensitizer, appears to play an important role not only in glucose and lipid metabolism but also in the development and progression of several obesity-related malignancies. In this review, we present recent findings on the association of adiponectin with several malignancies as well as recent data on underlying molecular mechanisms that provide novel insights into the association between obesity and cancer risk. We also identify important research questions that remain unanswered

Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation

Estimates of genomic heritability and genome-wide association study for fatty acids profile in Santa Inês sheep

Background: Despite the health concerns and nutritional importance of fatty acids, there is a relative paucity of studies in the literature that report genetic or genomic parameters, especially in the case of sheep populations. To investigate the genetic architecture of fatty acid composition of sheep, we conducted genome-wide association studies (GWAS) and estimated genomic heritabilities for fatty acid profile in Longissimus dorsi muscle of 216 male sheep. Results: Genomic heritability estimates for fatty acid content ranged from 0.25 to 0.46, indicating that substantial genetic variation exists for the evaluated traits. Therefore, it is possible to alter fatty acid profiles through selection. Twenty-seven genomic regions of 10 adjacent SNPs associated with fatty acids composition were identified on chromosomes 1, 2, 3, 5, 8, 12, 14, 15, 16, 17, and 18, each explaining ≥0.30% of the additive genetic variance. Twenty-three genes supporting the understanding of genetic mechanisms of fat composition in sheep were identified in these regions, such as DGAT2, TRHDE, TPH2, ME1, C6, C7, UBE3D, PARP14, and MRPS30. Conclusions: Estimates of genomic heritabilities and elucidating important genomic regions can contribute to a better understanding of the genetic control of fatty acid deposition and improve the selection strategies to enhance meat quality and health attributes

Methods for Monitoring Matrix-Induced Autophagy.

A growing body of research demonstrates modulation of autophagy by a variety of matrix constituents, including decorin, endorepellin, and endostatin. These matrix proteins are both pro-autophagic and anti-angiogenic. Here, we detail a series of methods to monitor matrix-induced autophagy and its concurrent effects on angiogenesis. We first discuss cloning and purifying proteoglycan fragment and core proteins in the laboratory and review relevant techniques spanning from cell culture to treatment with these purified proteoglycans in vitro and ex vivo. Further, we cover protocols in monitoring autophagic progression via morphological and microscopic characterization, biochemical western blot analysis, and signaling pathway investigation. Downstream angiogenic effects using in vivo approaches are then discussed using wild-type mice and the GFP-LC3 transgenic mouse model. Finally, we explore matrix-induced mitophagy via monitoring changes in mitochondrial DNA and permeability