An Intrinsically Disordered Region of the Acetyltransferase p300 with Similarity to Prion-Like Domains Plays a Role in Aggregation

Several human diseases including neurodegenerative disorders and cancer are associated with abnormal accumulation and aggregation of misfolded proteins. Proteins with high tendency to aggregate include the p53 gene product, TAU and alpha synuclein. The potential toxicity of aberrantly folded proteins is limited via their transport into intracellular sub-compartments, the aggresomes, where misfolded proteins are stored or cleared via autophagy. We have identified a region of the acetyltransferase p300 that is highly disordered and displays similarities with prion-like domains. We show that this region is encoded as an alternative spliced variant independently of the acetyltransferase domain, and provides an interaction interface for various misfolded proteins, promoting their aggregation. p300 enhances aggregation of TAU and of p53 and is a component of cellular aggregates in both tissue culture cells and in alpha-synuclein positive Lewy bodies of patients affected by Parkinson disease. Down-regulation of p300 impairs aggresome formation and enhances cytotoxicity induced by misfolded protein stress. These data unravel a novel activity of p300, offer new insights into the function of disordered domains and implicate p300 in pathological aggregation that occurs in neurodegeneration and cancer

YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion

Background: Genomic rearrangements involving the ETS family of transcription factors occur in 40–70 % of prostate cancer cases. ERG and ETV1 are the most common ETS members observed in these genetic alterations. The high prevalence of these rearrangements and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. Methods and Findings: We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewing’s Sarcoma. Since ERG and ETV1 belong to the same class of ETS factors as FLI1, we tested the ability of YK-4-279 to inhibit biological functions of ERG and ETV1 proteins in prostate cancer. YK-4-279 inhibited ERG and ETV1 mediated transcriptional activity in a luciferase assay. YK-4-279 also decreased ERG and ETV1 downstream target mRNA and protein expression in ETV1-fusion positive LNCaP and ERG fusion positive VCaP cells. YK-4-279 reduced the motility of LNCaP cells in a scratch assay and the invasive phenotype of both LNCaP and VCaP cells in a HUVEC invasion assay. Fusion-negative PC3 cells were unresponsive to YK-4-279. SiRNA mediated ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Concurrently, transient ERG expression in PC-3 cells resulted in increased invasive potential, which was reduced by YK-4-279. Conclusion: These data demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostat

High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo.Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy

The emerging role of insulin-like growth factor 1 receptor (IGF1r) in gastrointestinal stromal tumors (GISTs)

Recent years have seen a growing interest in insulin-like growth factor 1 receptor (IGF1R) in medical oncology. Interesting data have been reported also on IGF1r in gastrointestinal stromal tumors (GISTs) especially in children and in young adult patients whose disease does not harbour mutations on KIT and PDGFRA and are poorly responsive to conventional therapies. However, it is too early to reach conclusions on IGF1R as a novel therapeutic target in GIST because the receptor's biological role is still to be defined and the clinical significance in patients needs to be studied in larger studies. We update and comment the current literature on IGF1R in GISTs and discuss the future perspectives in this promising field

PI3K/AKT is involved in mediating survival signals that rescue Ewing tumour cells from fibroblast growth factor 2-induced cell death

While in vitro studies had shown that fibroblast growth factor 2 (FGF2) can induce cell death in Ewing tumours, it remained unclear how Ewing tumour cells survive in vivo within a FGF2-rich microenvironment. Serum- and integrin-mediated survival signals were, therefore, studied in adherent monolayer and anchorage-independent colony cell cultures. In a panel of Ewing tumour cell lines, either adhesion to collagen or exposure to serum alone only had a minor protective effect against FGF2. However, both combined led to complete resistance to 5 ng ml−1 FGF2 in three of four FGF2-sensitive cell lines (RD-ES, RM-82 and WE-68), and to an increased survival as compared to other culture conditions in TC-71 cells. Inhibition studies with LY294002 demonstrated that the serum signal is mediated via the phosphoinositide 3-OH kinase/AKT pathway. Thus, Ewing tumour cells escape FGF2-induced cell death by modulating FGF2 signalling. The tumour microenvironment provides the necessary survival signals by integrin-mediated adhesion and soluble serum factor(s). These survival signals warrant further investigation as a potential resistance mechanism to other apoptosis-inducing agents in vivo

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation

IGF1 Is a Common Target Gene of Ewing's Sarcoma Fusion Proteins in Mesenchymal Progenitor Cells

The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis

Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma

IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT–PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins

R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt

IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling