Diffuse intrinsic pontine glioma (DIPG) and pediatric glioblastoma (pGBM) are heterogeneous brain tumors characterized by different anatomical and molecular subgroups and the presence of genetically and phenotypically distinct subclonal cell populations. It is recognized that exosomes mediate cross-talk among tumor cells. We hypothesize that there are different exosome-mediated paracrine signaling promoting tumour progression in DIPG and pGBM. Our aim was to determine the specific DIPG and pGBM-derived exosome oncogenic signatures. We used a panel of fifteen patient primary-derived cell lines, which included nine DIPG (seven H3.3 K27M, one H3.3 K27M/ACVR1 and one H3.1 K27M/ACVR1), one diffuse midline glioma H3.3 K27M and three GBM (one H3.3 G34R and two histone WT). Conditioned medium was collected from cells maintained under stem-cell culture condition, adherent on laminin and/or as neurospheres (NS), and exosomes harvested through serial centrifugations. Electron microscopy demonstrated that the isolated microvescicles are exosomes sized between 50–80 nm. DIPG derived-exosomes appeared to have a variable cargo of total protein (µg)/106 cells, which was higher than for pGBM-exosomes. Proteomic analysis revealed that proteins associated with vesicle docking, exocytosis and synaptic transmission were exclusively enriched in pontine-derived exosomes, while cell-cell and cell-matrix interaction proteinswere exclusive tohemispheric ones. Proteins in common to the two locations were involved in metabolism and energy pathways. Interestingly, principle component analysis on the different molecular subgroups suggests that ACVR1 may be not implicated in the exosomal proteomic signature. Exosomal miRNA profile appeared to be driven by the two main histone mutated subgroups H3.3 K27M and H3.1 K27M with the latter overexpressing hypoxia and angiogenic-associated miRNAs, leading to distinct oncogenic programs with different specific potential therapeutic targets. This study aimed to development new diagnostic/prognostic tools for DIPG and pGBM patients. Further investigations are aimed to identify new therapeutic strategies to inhibit the cross-talk among glioma subpopulations.
Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.
Abstract Morgana is a ubiquitous HSP90 co-chaperone protein coded by the CHORDC1 gene. Morgana heterozygous mice develop with age a myeloid malignancy resembling human atypical myeloid leukemia (aCML), now renamed MDS/MPN with neutrophilia. Patients affected by this pathology exhibit low Morgana levels in the bone marrow (BM), suggesting that Morgana downregulation plays a causative role in the human malignancy. A decrease in Morgana expression levels is also evident in the BM of a subgroup of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients showing resistance or an incomplete response to imatinib. Despite the relevance of these data, the mechanism through which Morgana expression is downregulated in patients’ bone marrow remains unclear. In this study, we investigated the possibility that Morgana expression is regulated by miRNAs and we demonstrated that Morgana is under the control of four miRNAs (miR-15a/b and miR-26a/b) and that miR-15a may account for Morgana downregulation in CML patients.
Chaperones and scaffold proteins are key elements involved in controlling the assembly of molecular complexes required for coordinated signal transduction. Here we describe morgana and melusin, two phylogenetically conserved chaperones that cooperate with Hsp90 and regulate signal transduction in important physiopathological processes. While morgana is ubiquitously expressed, melusin expression is restricted to striated muscles. Despite high sequence homology, the two chaperones have distinct functions. Morgana controls genomic stability by regulating the centrosome cycle via ROCKII kinase. Melusin, however, organizes ERK signal transduction in cardiomyocytes and regulates cardiac compensatory hypertrophy in response to different stress stimuli.
The epigenetic repressor BMI1 plays an integral role in promoting the self-renewal and proliferation of many adult stem cell populations, and also tumor types, primarily through silencing the Cdkn2a locus, which encodes the tumor suppressors p16
Abstract Malignant melanoma is one of the most aggressive human cancers, with a high potential for lethal metastasis and therapeutic resistance. The epigenetic chromatin regulator Bmi1 acts as a key component of the Polycomb Repressor Complex 1 (PRC1) and is a known oncogene. Various studies shown that BMI1 is highly expressed in many human hematopoietic malignancies and in different solid tumor types including prostate, breast, ovarian, colon, brain and lung cancer. Importantly, high Bmi1 expression is an excellent predictor of both metastatic progression and poor therapeutic response. Bmi1's role in tumor onset has been assessed using germline mutant mice showing that Bmi1 ablation efficiently suppress tumor development. This impaired tumorigenicity correlates with an impairment of the self-renewal and proliferative properties of tumor initiating cells, mainly due to Bmi1's repressive activity on the Ink4a/Arf tumor suppressor locus. Nevertheless, this function does not explain why Bmi1 is frequently upregulated in tumors nor Bmi1's role in tumor progression. To evaluate Bmi1's contribution to melanoma progression, we upregulated Bmi1 in two different melanoma cell lines, a murine metastatic cell line and a poorly metastatic human cell line. We found that Bmi1 overexpression activates or increases invasive behavior of both cell lines, without enhancing their proliferative capacity. This finding allowed us to dissect Bmi1's oncogenic activity at later stages of tumorigenesis, in the absence of Bmi1's deregulation of cell proliferation. Our data show that Bmi1 significantly enhances in vitro cell movement and modulates adhesion. Bmi1 also protects cells from apoptosis induced by different stimuli and increases lung metastasis formation in vivo. To characterize Bmi1-induced changes in gene expression, we have performed comparative deep sequencing of total RNA libraries generated from Bmi1 or GFP overexpressing cells. Gene ontology analysis revealed enrichment in cancer-relevant pathways that seem to be controlled by Bmi1, including cell movement, cell death and survival, tissue development and cell morphology. In particular, Bmi1 expression modulates the gene regulatory programs associated with EMT, TGFβ and the non-canonical Wnt pathways. We have identified a new Bmi1 target gene, poorly characterized, that seems to be responsible for Bmi1-induced melanoma progression. These data determine a causal role for Bmi1 in melanoma cell migration and establishment of distant melanoma metastasis. Citation Format: Roberta Ferretti, Jacqueline A. Lees. Molecular mechanism driving BMI1-induced melanoma metastasis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C11.
Abstract Pediatric high-grade gliomas (pHGG) are heterogeneous brain tumors for which new specific diagnostic/prognostic biomarkers are needed. In this study, we aimed to identify new pHGG subgroup specific biomarkers by exploiting exosomes, known vehicles of oncogenic signals. We used plasma from 23 patients (including 6 controls) and conditioned medium from 12 patient-derived cell-lines, representing all locational and molecular subgroups. Upon exosome isolation, total RNA was extracted and miRNAs were assessed using a PCR Panel. Analysis of plasma miRNome showed that tumor exosomal samples were largely clustered together, independently from their locational and/or molecular subgroup. We identified 20 significantly upregulated and 25 downregulated miRNAs compared to controls. Interestingly, 27 miRNAs were expressed only in tumors. Furthermore, the unsupervised clustering showed a clear separation based on locational (hemispheric vs pontine) and mutational (WT vs H3.3G34R or H3.3G34R vs H3K27M) subgroup comparisons, with the identification of distinct miRNomes underlying the key role of location and mutations in defining the pHGG exosomal miRNA profile. This was further confirmed analyzing the miRNOme from cell-line derived exosomes. Moreover, we identified a pool of significantly differentially regulated miRNAs in diagnose vs relapse and biopsy vs autopsy cell-lines. Most importantly, when comparing hemispheric vs pontine and H3.3G34R vs H3.3K27M, we identified respectively four and three miRNas equally dysregulated and in common between plasma and cell-lines. Those were strongly associated mainly to transcriptional regulation and targeting TTC9, linked to cancer invasion and metastasis. Based on this, we suggest exosomal miRNAs as a powerful new pHGG diagnostic/prognostic tool.
Melusin is a muscle specific protein required for heart hypertrophy in response to mechanical overload. Here we describe a protein 63% homologous to melusin, named chp‐1, expressed in all tissues tested, including muscles, and highly conserved from invertebrates to human. Both proteins are characterized in their N‐terminal half by a tandemly repeated zinc binding 60 amino acid domain with a motif of uniquely spaced cysteine and histidine residues. These motives are highly conserved from plants to mammals and have been recently named CHORD (for cysteine and histidine rich domain) domains. At the C‐terminal end melusin contains a calcium binding stretch of 30 acidic amino acid residues which is absent in chp‐1. While invertebrate genome contains only one gene coding for a chp‐1 homolog, two genes coding for CHORD containing proteins (chp‐1 and melusin) are present in vertebrates. Sequence analysis suggests that the muscle specific CHORD containing protein melusin originated by a gene duplication event during early chordate evolution.
