Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

Gene expression profile of human lymphocytes exposed to (211)At alpha particles.

In this study, the Whole Human Genome 44K DNA microarray assay was used for the first time to obtain gene expression profiles in human peripheral blood lymphocytes 2 h after exposure (in suspension) to 6.78 MeV mean energy alpha particles from extracellular (211)At. Lymphocytes were exposed to fluences of 0.3-9.6 x 10(6) alpha particles/cm(2) [corresponding to mean absorbed alpha-particle doses (D(alpha)) of 0.05-1.60 Gy] over 30 min. Significantly modulated expression was identified in 338 early-response genes. Up-regulated expression was evident in 183 early-response genes, while the remaining 155 were down-regulated. Over half of the up-regulated genes and 40% of the down-regulated genes had a known biological process related primarily to cell growth and maintenance and cell communication. Genes associated with cell death were found only in the up-regulated genes and those with development only in the down-regulated genes. Eight selected early-response genes that displayed a sustained up- or down-regulation (CD36, HSPA2, MS4A6A, NFIL3, IL1F9, IRX5, RASL11B and SULT1B1) were further validated in alpha-particle-irradiated lymphocytes of two human individuals using the TaqMan(R) RT-qPCR technique. The results confirmed the observed microarray gene expression patterns. The expression modulation profiles of IL1F9, IRX5, RASL11B and SULT1B1 genes demonstrated similar trends in the two individuals studied. However, no significant linear correlation between increasing relative gene expression and the alpha-particle dose was evident. The results suggest the possibility that a panel of genes that react to alpha-particle radiation does exist and that they merit further study in a greater number of individuals to determine their possible value regarding alpha-particle biodosimetry.

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis.

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO(2), whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC-MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼600 individual proteins) and quantified proteins (>95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins.

Isotope coded protein label quantification of serum proteins--comparison with the label-free LC-MS and validation using the MRM approach.

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids - serum - and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM based protein quantification technique. Regarding the ICPL approach, serum samples used in this study were depleted of high abundant proteins, labeled with ICPL and fractionated according to their respective pI (3-5, 5-7 and 7-12). The samples were further subjected to tryptic digestion followed by treatment with the Glu-C enzyme. The peptides were analyzed on a 2D-nano-LC system using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The LC system was connected on-line with the electrospray ion-trap mass spectrometer. For the label-free quantification the serum samples were depleted and digested with trypsin. A proteome-wide comparison was performed using highly reproducible LC and data independent alternate scanning in conjunction with a high mass accuracy orthogonal time-of-flight mass spectrometer. Selected proteins, found by both methods, were validated using the MRM approach. For this purpose non-depleted tryptically digested serum samples were analyzed by LC coupled with a triple-quadrupole MS. The relative protein quantification using ICPL and mass spectrometry allowed for the detection of approximately 200 proteins, whereas about 2/3 of those contained the ICPL label and could therefore be quantified. Label-free approach used no fractionation, less sample and was able to identify and quantify over 110 proteins. The identified proteins covered generally 3-4 orders of magnitude of protein concentration in human serum. Changes in relative abundance of eight proteins were validated using MRM. This study, for the first time, shows the ability of the relative protein quantification based upon ICPL and 2D-LC-MS/MS to quantify serum biomarkers. It provides two additional label-free approaches that could validate and bring additional value to the label-based results, offering a starting point for comprehensive proteomics studies aiming at revealing biomarkers of clinical relevance.

[Targeted therapy: toward a clean and effective war against cancer]. / Le ciblage thérapeutique: vers une guerre propre et efficace contre le cancer.

One promising avenue towards the development of more selective, better anticancer drugs consists in the targeted delivery of bioactive compounds to the tumor environment by means of binding molecules specific for tumor-associated biomarkers. Eligibility of such markers for therapeutic use implies ideally three criteria : (i) accessibility from the bloodstream, (ii) expression at sufficient level and (iii) no (or much lower) expression in normal tissues. Most current discovery strategies (such as biomarker searching into body fluids) provide no clue as to whether proteins of interest are accessible, in human tissues, to suitable high-affinity ligands, such as systemically delivered monoclonal antibodies. Innovative proteomic technologies are able to identify such accessible biomarkers and represent a key step in the clinical development of such target therapies.

Early gene expression in human lymphocytes after gamma-irradiation-a genetic pattern with potential for biodosimetry.

PURPOSE: Identification of early radiation response genes (ERG) in human lymphocytes after gamma-irradiation by using the whole-human-genome DNA-microarrays and the evaluation of their possible role in rapid radiation biodosimetry by applying real-time quantitative polymerase chain reaction (RT-qPCR) methodology for validation in a small group of human individuals. MATERIALS AND METHODS: Whole blood from a healthy human donor was exposed at 37 degrees C to 137Cs gamma-radiations (absorbed dose: 1-4 Gy). Fifteen minutes following irradiation the lymphocytes were isolated from the blood (for 2 h at 20 degrees C) and their gene expression was investigated using the DNA-microarrays. Subsequently, 14 genes were selected and validated using the TaqMan probes based upon the RT-qPCR assay within a group of 6 human donors. RESULTS: A dose-related relative change in quantitative gene expression using the DNA-microarray assay was demonstrated in 24 of 102 genes. Up-regulation of expression was observed in 15 genes: CD69 (CD69 molecule), CDKN1A (cyclin-dependent kinase inhibitor 1A), EGR1 (early growth response 1), EGR4 (early growth response 4), FLJ35725 (chromosome 4 ORF 23), hCG2041177 (hCG - human Celera Genome), hCG1643466.2, IFN-gamma (interferon-gamma), ISG20L (interferon stimulated exonuclease gene 20 kDa - like 1), c-JUN (jun oncogene), MDM2 (mouse double minute 2), MUC5B (mucine), PLK2 (polo-like kinase 2), RND1 (rho-family GTPase 1) and TNFSF9 (tumour necrosis factor superfamily member 9). Down-regulation of expression was found in the remaining nine genes: GRIK3 (glutamate receptor ionotropic kainate 3), hCG1985174, hCG1998530, hCG2038519, OCLN (occludin), RPL10A (ribosomal protein L10a), SERHL2 (serine hydrolase-like 2), SGK3 (serum/glucocorticoid regulated kinase 3) and STARD13 (START domain containing 13). CONCLUSION: A significant correlation between absorbed radiation dose and change in relative gene expression was particularly evident for EGR1, EGR4, IFN-gamma, c-JUN and TNFSF9 (p < or = 0.05). Results warrant the further investigation of these ERG as potential biodosimetric markers.