WO2022152698A1 - Use of npdk-d to evaluate cancer prognosis - Google Patents

Use of npdk-d to evaluate cancer prognosis Download PDF

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WO2022152698A1
WO2022152698A1 PCT/EP2022/050446 EP2022050446W WO2022152698A1 WO 2022152698 A1 WO2022152698 A1 WO 2022152698A1 EP 2022050446 W EP2022050446 W EP 2022050446W WO 2022152698 A1 WO2022152698 A1 WO 2022152698A1
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cancer
ndpk
protein
expression
cells
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PCT/EP2022/050446
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French (fr)
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Marie-Lise Lacombe
Mathieu BOISSAN
Uwe SCHLATTNER
Malgorzata TOKARSKA-SCHLATTNER
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Assistance Publique - Hôpitaux De Paris
Sorbonne Universite
Université Grenoble Alpes
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Publication of WO2022152698A1 publication Critical patent/WO2022152698A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds

Definitions

  • the invention relates to a method for predicting the survival time of a patient suffering from a cancer comprising determining the expression level of NDPK-D and to the treatment of cancer in a subject in need thereof by using the NDPK-D protein or a fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.
  • Carcinomas the most prevalent malignancies in humans, arise from normal epithelial tissues in a multistep progression from benign precursor lesions. Metastasis, the final step in malignancy, is the cause of death for more than 90% of cancer patients. Molecular mechanisms underlying metastasis have to be elucidated for accurate detection and treatment (1).
  • metastatic disease complex pathways involving the tumor cell and the microenvironment mediate tumor invasion at the primary site, survival and arrest in the bloodstream, extravasation, and colonization at a secondary site.
  • the first step in the metastatic cascade i.e. the breakdown of epithelial intercellular adhesion and the acquisition of an invasive program, allows epithelial cancer cells to breach the basement membrane and to invade stromal type I fibrillar collagen.
  • epithelial-mesenchymal transition EMT
  • epithelial cells lose some of their epithelial characteristics, including cell adhesion and cell polarity; cytoskeletal rearrangement occurs that ultimately leads to an increased motility and an invasive phenotype.
  • NME1/NM23-H1 encodes the nucleoside diphosphate kinase A (NDPK-A), which catalyzes synthesis of nucleoside triphosphates including GTP from corresponding nucleoside diphosphates and ATP.
  • NDPK-A nucleoside diphosphate kinase A
  • ten isoforms of the NME/NM23/NDPK family have been identified (reviewed in (4)), among those, the mitochondrial isoform NME4/NM23-H4, also called NDPK-D (5, 6). If NME1 has a well-known anti-metastatic activity, the contribution of the other isoforms including NME2 and NME3 is much less documented or even unknown for NME4.
  • NME4/NM23-H4 is a mitochondrial enzyme, which binds to the mitochondrial inner membrane (MIM) through anionic phospholipids, mainly cardiolipin (CL), and is principally oriented towards the intermembrane space (5-7).
  • the enzyme acts as a lipid-dependent mitochondrial switch with dual function: (i) in phosphotransfer serving, in particular, for local GTP supply to the mitochondrial dynamin GTPase, Optic Atrophy 1 (OPA1), a driver of mitochondrial fusion (8, 9) and (ii) in CL transfer from the inner to the outer membrane, where this phospholipid serves as pro-mitophagic or pro- apoptotic signal (8, 10).
  • HeLa cells which naturally express low levels of NDPK-D, were stably transfected with expression vectors, either empty or designed to express NDPK-D wild type or mutant proteins.
  • Single point mutations were chosen to suppress either the catalytic NDPK activity of the enzyme or its ability to bind CL, which localizes the enzyme to the inner membrane and is essential for its function in CL intermembrane transfer. Both mutations led to similar alterations in the cellular behavior, linked to altered mitochondrial structure and function, reprogramming of protein expression, and a morphotypic switch towards a pro- metastatic phenotype.
  • Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. In human cancer, NME4 expression is negatively associated with tumor aggressiveness, and is a good prognosis factor for beneficial clinical outcome.
  • the invention relates to a method for predicting the survival time of a patient suffering from a cancer comprising determining the expression level of NDPK-D and to the treatment of cancer in a subject in need thereof by using the NDPK-D protein or a fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.
  • the invention is defined by its claims.
  • a first aspect of the invention relates to a method for predicting the survival time of a patient suffering from a cancer, comprising i) determining the expression level of NDPK-D in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is higher than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is lower than its predetermined reference value.
  • Another aspect of the invention relates to a method for predicting the invasiveness of a cancer, comprising i) determining in a sample obtained from the patient the expression level of NDPK-D ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is higher than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is lower than its predetermined reference value.
  • the cancer may be a solid or a liquid cancer.
  • the cancer is a solid cancer.
  • the cancer is a metastatic cancer.
  • the cancer is a solid metastatic cancer.
  • the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular
  • the cancer may be selected in the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, uterine cancer, esophageal cancer, pheochromocytoma, paraganglioma and sarcoma.
  • the cancer may be selected in the group consisting of breast carcinoma, ovarian serous cystadenocarcinoma, lung carcinoma, pancreatic ductal adenocarcinoma, uterine corpus endometrial carcinoma, esophageal squamous cell carcinoma, pheochromocytoma, paraganglioma and sarcoma.
  • the sample can be blood, peripheral -blood, serum, plasma, tumoral circulating cells, tumor sample that is to say a sample obtained from the tumor or a biopsy obtained from the tumor or mitochondria purified from the tumor.
  • the sample is a solid tumor sample.
  • the sample can also arise from the uninvolved corresponding tissue sample.
  • the term “subject” or “patient” denotes a mammal.
  • a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with a cancer, particularly a metastatic cancer.
  • NDPK-D for “nucleoside diphosphate kinase-D”, also known as NME4/NM23-H4, is a ubiquitous mitochondrial enzyme that catalyzes transfer of gammaphosphates, via a phosphohistidine intermediate, between nucleoside and deoxynucleoside tri- and diphosphates.
  • NME4/NM23-H4 is a ubiquitous mitochondrial enzyme that catalyzes transfer of gammaphosphates, via a phosphohistidine intermediate, between nucleoside and deoxynucleoside tri- and diphosphates.
  • the Gene sequence its Entrez Gene reference number is: 4833; for the protein, the UniProt reference number is: 000746 and NCBI reference is: NP 005000.1; for the mRNA, the GenBank reference is: Y07604.1.
  • survival time denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as pancreatic cancer (according to the invention).
  • the survival time rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.
  • survival time can regroup the term “Overall survival (OS)”.
  • OS denotes the time from diagnosis of a disease such as cancer (according to the invention) until death from any cause.
  • the overall survival rate is often stated as a two-year survival rate, which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.
  • the term “invasiveness of a cancer” is the capacity of a cancer to lead to the formation of metastases.
  • Measuring the expression level of NDPK-D can be done by measuring the gene expression level of NDPK-D or by measuring the level of the protein NDPK-D and can be performed by a variety of techniques well known in the art.
  • the expression level of a gene may be determined by determining the quantity of mRNA.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence or mRNA) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • catalysts such as enzymes
  • haptens that can be detected by antibody binding interactions
  • paramagnetic and magnetic molecules or materials paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrromethene boron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOT® (obtained, for example, from Life Technologies (Quantum Dot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the band gap of the semiconductor material used in the semiconductor nanocrystal.
  • This emission can he detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif.).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can be used in a metallographic detection scheme.
  • SISH silver in situ hybridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following nonlimiting examples: biotin, digoxigenin, DNP (dinitrophenol), and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following nonlimiting examples: biotin, digoxigenin, DNP (dinitrophenol), and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophy
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acid sequences (e.g., target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can be labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 nm).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • the level of NDPK-D proteins may also be measured and can be performed by a variety of techniques well known in the art.
  • techniques like ELISA see below allowing to measure the level of the soluble proteins are particularly suitable.
  • the sample is solubilized since NDPK-D is bound to the mitochondrial membrane.
  • the “level of protein” or the “protein level expression” or the “protein concentration” means the quantity or concentration of said protein.
  • the “level of protein” means the level of NDPK-D protein fragments.
  • the “level of protein” means the quantitative measurement of NDPK-D protein expression relative to a negative control.
  • the proteins in the sample or mitochondria isolated thereof are solubilized with detergent, since NDPK-D is bound to the mitochondrial membrane.
  • the proportion of NDPK-D that remains in the particulate fraction without detergent treatment and that is above a predetermined reference value would relate to membrane-bound protein necessary for metastasis-suppression function. It would therefore have predictive value.
  • enzymatic activity of NDPK-D is measured in mitochondria isolated form tumour tissue.
  • protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • ELISA ELISA
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresismass spectroscopy technique (CE-MS).
  • the reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • Methods of the invention may comprise a step consisting in comparing the proteins and fragments concentration in circulating cells with a control value.
  • concentration of protein refers to an amount or a concentration of a transcription product, for instance the protein NDPK-D.
  • a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • relative units can be employed to describe a concentration.
  • concentration of proteins may refer to fragments of the protein NDPK-D.
  • fragments of NDPK-D protein may also be measured.
  • Predetermined reference values used for comparison of the expression levels may comprise “cut-off’ or “threshold” values that may be determined as described herein.
  • Each reference (“cut-off’) value for NDPK-D level may be predetermined by carrying out a method comprising the steps of: a) providing a collection of samples from patients suffering of a cancer and/or samples of the corresponding uninvolved tissues as described in the invention; b) determining the level of NDPK-D for each sample contained in the collection provided at step a); c) ranking the tumor tissue samples according to said level d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level, e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient; f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve; g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets
  • the expression level of NDPK-D has been assessed for 100 cancer samples of 100 patients.
  • the 100 samples are ranked according to their expression level.
  • Sample 1 has the highest expression level and sample 100 has the lowest expression level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
  • the reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.
  • the reference value (cut-off value) may be used in the present method to discriminate cancer samples and therefore the corresponding patients.
  • Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.
  • kits for performing the methods of the invention comprise means for measuring the expression level of NDPK-D in the sample obtained from the patient.
  • kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled.
  • probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers that may be prelabelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the present invention also relates to NDPK-D as biomarkers for outcome of cancer patients.
  • the present invention also relates to NDPK-D as biomarkers of invasiveness of cancer.
  • the invention relates to the NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression for use in the treatment of cancer in a subject in need thereof.
  • NDPK-D protein or fragment thereof or a fusion protein thereof or the agent for NDPK-D protein expression will be useful to treat metastasis.
  • the invention also relates to a method for treating a cancer in a subject in need thereof comprising the administration to said subject the NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.
  • an agent for NDPK-D protein expression denotes an agent which can increase or restore the NDPK-D protein expression or increase the activity of the NDPK-D.
  • the agent can also increase or restore the NDPK-D gene expression.
  • the agent for NDPK-D protein expression is a vector encoding NME4, NME4 mRNA or is the NME4 mRNA molecule itself.
  • the NME4 mRNA molecule may be included in an appropriate vector.
  • the appropriate vector may be a membrane or lipid vesicle.
  • the cancer may be a solid or a liquid cancer.
  • the cancer is a solid cancer.
  • the cancer is a metastatic cancer.
  • the cancer is a solid metastatic cancer.
  • the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular
  • the cancer may be selected in the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, uterine cancer, esophageal cancer, pheochromocytoma, paraganglioma and sarcoma.
  • the cancer may be selected in the group consisting of breast carcinoma, ovarian serous cystadenocarcinoma, lung carcinoma, pancreatic ductal adenocarcinoma, uterine corpus endometrial carcinoma, esophageal squamous cell carcinoma, pheochromocytoma, paraganglioma and sarcoma.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder (like cancer) or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the NDPK-D protein of the invention is an isolated, synthetic or recombinant NDPK-D protein.
  • said NDPK-D protein comprises a sequence as set forth by SEQ ID NO: 1:
  • the protein of the present invention comprises or consists of an amino acid sequence having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % of identity with the SEQ ID NO: 1.
  • a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100% of identity with the second amino acid sequence.
  • Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).
  • the polypeptide of the invention is a functional conservative variant of the polypeptide according to the invention.
  • a “function-conservative variant” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a “function-conservative variant” also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent polypeptide to which it is compared.
  • a further aspect of the present invention relates to a fusion protein comprising the protein according to the invention that is fused to at least one heterologous polypeptide.
  • the invention relates also to a fusion protein which consist to the protein according to the invention fused to at least one heterologous polypeptide.
  • fusion protein refers to the protein or peptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.
  • the fusion protein comprises the protein or peptide that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.
  • the term “directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the protein or peptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.
  • the last amino acid of the C-terminal end of said protein or peptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said protein or peptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.
  • spacer refers to a sequence of at least one amino acid that links the protein or peptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances.
  • the heterologous polypeptide is a cell -penetrating peptide, a Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide or a membranous penetrating sequence.
  • TAT Transactivator of Transcription
  • cell-penetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT, mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
  • the proteins, peptides or fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides or fusion proteins, by standard techniques for production of amino acid sequences.
  • polypeptides or fusion proteins of the invention can be synthesized using recombinant DNA techniques as is now well-known in the art.
  • these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well- known techniques.
  • the proteins, peptides, fusion proteins or the NME4 mRNA molecule itself of the invention can be used in an isolated (e.g., purified) form or contained in a vehicle, such as a membrane or lipid vesicle (e.g. a liposome).
  • a vehicle such as a membrane or lipid vesicle (e.g. a liposome).
  • proteins, polypeptides or fusion proteins according to the invention may be modified in order to improve their therapeutic efficacy and their stability using well-known techniques.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • a strategy for improving drug stability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • PEGylation i.e. covalent attachment of one or more polyethyleneglycol (PEG) groups
  • PEG polyethyleneglycol
  • the benefits include among others: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles et.al., 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide.
  • the proteins, peptides or fusion proteins of the invention may be covalently linked with one or more PEG group(s).
  • PEG group(s) One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc.
  • the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH ("methoxy PEG").
  • a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called branched PEGs.
  • Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythritol, and sorbitol.
  • a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide.
  • One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., 1995).
  • the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N- hydroxysuccinimide (NHS), aldehyde, nitrophenyl carb onate (NPC), and tresylate (TRES)).
  • Suitable activated polymer molecules are commercially available, e.
  • the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90/13540.
  • activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference).
  • activated PEG polymers include the following linear PEGs : NHS-PEG (e g.
  • SPA-PEG SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG
  • NOR-PEG BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.
  • the conjugation of the proteins, peptides or fusion proteins and the activated polymer molecules is conducted by use of any conventional method. Conventional methods are known to the skilled artisan. The skilled person will be aware that the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).
  • the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).
  • polypeptides are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH2), C (for SH if at least one cysteine is conserved) or/and Q and N (for the amide function).
  • additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue.
  • additional PEGylation sites are chemically introduced by modifying amino acids on polypeptides of the invention.
  • PEGs are conjugated to the polypeptides or fusion proteins through a linker.
  • Suitable linkers are well known to the skilled person.
  • a preferred example is cyanuric chloride ((Abuchowski et al., 1977); US 4,179, 337).
  • the pegylated polypeptides provided by the invention have a serum half-life in vivo of at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified polypeptide.
  • the agent for NDPK-D protein expression of the invention is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for NDPK-D protein, a nucleic acid sequence encoding for the fusion protein, a nucleic acid encoding a fragment of a NDPK-D protein, a cell expressing NDPK-D protein, and agent inducing NDPK-D gene expression and their combinations.
  • said nucleic acid encoding for NDPK-D protein comprises a sequence as set forth by SEQ ID NO: 2.
  • the nucleic acid encoding for NDPK-D protein for example comprises or consists of a sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % identical to sequence SEQ ID NO: 2.
  • a sequence "encoding" an expression product such as a RNA, polypeptide, protein, or enzyme
  • a sequence "encoding" an expression product is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
  • a coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
  • nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art.
  • said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
  • a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a polypeptide or a fusion protein of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted.
  • suitable elements for controlling transcription in particular promoter, enhancer and, optionally, terminator
  • recombinant vectors may, for example, be cloning vectors, or expression vectors.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • a DNA or RNA sequence e.g. a foreign gene
  • Any expression vector for animal cell can be used.
  • suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al., 1984), pKCR (O'Hare et al., 1981), pSGl beta d2-4 (Miyaji et al., 1990) and the like.
  • plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
  • viral vectors include adenoviral, lentiviral, retroviral, herpes virus and AAV vectors.
  • recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • virus packaging cells include PA317 cells, Psi CRIP cells, GPenv+ cells, 293 cells, etc.
  • promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana et al., 1987), promoter (Mason et al., 1985) and enhancer (Gillies et al., 1983) of immunoglobulin H chain and the like.
  • a further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a protein or a fusion protein according to the invention or a vector according to the invention.
  • a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention.
  • transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • a host cell that receives and expresses introduced DNA or RNA has been "transformed”.
  • prokaryotic cells for expressing and producing proteins, peptides or fusion proteins of the invention, prokaryotic cells, in particular E. coli cells, will be chosen.
  • it is not mandatory to produce the polypeptide or the fusion protein of the invention in a eukaryotic context that will favour post-translational modifications (e.g. glycosylation).
  • prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins.
  • typical eukaryotic cell lines such as CHO, BHK-21, COS-7, C127, PER.C6, YB2/0, HEK293, mononuclear macrophage/monocyte-lineage hematopoietic precursors, Haematopoietic stem cells, Mononuclear precursor cells, osteoblast or inactive osteoclast could be used, for their ability to process to the right post-translational modifications of the fusion protein of the invention.
  • the construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques.
  • the protein, peptide or the fusion protein of the invention can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the polypeptide or the fusion protein expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.
  • a further aspect of the invention relates to a method for producing a protein, peptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said protein, peptide or fusion protein; and (ii) recovering the expressed protein, peptide or fusion protein.
  • the agent for NDPK-D protein expression of the invention is an agent inducing NDPK-D gene and peptide expression selected from the group consisting of, but not limited to, Human Cytomegalovirus (HCMV), VHL/E HCMV strain, and TB40/E HCMV strain.
  • HCMV Human Cytomegalovirus
  • VHL/E HCMV strain VHL/E HCMV strain
  • TB40/E HCMV strain TB40/E HCMV strain.
  • the invention relates to an anti-cancer agent for use in the treatment of a cancer in a patient with a bad prognosis as described above.
  • the invention also relates to the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention for use in the treatment of a cancer in a patient with a bad prognosis as described above.
  • the invention relates to an anti-cancer agent in combination with the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention for use in the treatment of a cancer in a patient with a bad prognosis as described above.
  • the invention also relates to a method for treating a cancer in a patient with a bad prognosis as described above comprising the administration to said patient of an anti-cancer agent.
  • the invention also relates to a method for treating a cancer in a patient with a bad prognosis as described above comprising the administration to said patient of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention.
  • Anti-cancer agent can be selected in the group consisting in cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin,
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional anti-cancer agent may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • additional therapeutic active agent can be added like an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron.
  • the further therapeutic active agent can be an hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or nonopioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
  • the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • CTL4 cytotoxic T lymphocyte-associated protein 4
  • PDCD1 programmed cell death 1
  • NK cells like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • the checkpoint blockade cancer immunotherapy agent is an antibody.
  • the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
  • the invention also relates to a pharmaceutical composition comprising A NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression for use in the treatment of a cancer in subject in need thereof.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-cancer treatment for use in the treatment of cancer in a subject with a bad prognosis as described above.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention as described above or the anti -cancer agent according to the invention are administered to the subject in a therapeutically effective amount.
  • a therapeutically effective amount of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention as above described or the anti-cancer agent is meant a sufficient amount of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent for treating cancer at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or anti-cancer agent will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anticancer agent employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent employed; the duration of the treatment; drugs used in combination or coincidental with the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent, preferably from 1 mg to about 100 mg of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention or the anti-cancer agent may be used in a concentration between 0.01 pM and 20 pM, particularly, the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the invention or the anti-cancer agent may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM.
  • the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent is administered to the subject in the form of a pharmaceutical composition.
  • the invention also relates to a therapeutic composition comprising the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or an anti-cancer agent for use in the treatment of a cancer in a subject in need thereof.
  • the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients such as biodegradable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Solutions comprising the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the invention or the anti-cancer agent of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
  • NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti -cancer agent of the invention plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Migration, invasion, and MMP activity of MDA-MB-231 cells genetically modified for NDPK-D and of ZR75-1 cells depleted for NDPK-D.
  • B Type I collagen invasion assay of MDA-MB-231 cells.
  • FIG. 2 Adhesion properties of MDA-MB-231 cells genetically modified for NDPK-D and of ZR75-1 cells depleted for NDPK-D.
  • A Two MDA-MB-231 clones were analyzed for each conditions named as described in the legend to Figure 1. The size of the aggregates observed is depicted as the area of their horizontal projections. Data show means ⁇ SEM of three independent biological replicates imaged. *****p ⁇ 0.00001 relative to control/empty vector (CTR).
  • CTR control/empty vector
  • B Two different siRNA targeting NME4 were used for ZR75-1 cell aggregation assay. The size of the aggregates observed in light microscopy representative images is depicted as the area of their horizontal projections. Data show means ⁇ SEM of three independent biological replicates imaged. *****p ⁇ 0.00001 relative to scramble control (Scr).
  • FIG. 3 NME4-related metastasis-suppression.
  • Experimental metastasis assay where two HeLa clones for each condition, empty control vector (CTR), wild-type (WT), and the kinase-dead mutant (KD), were injected in the tail vein of nude mice. After 13 weeks, mice were sacrificed, lungs removed, and the number of lung metastases counted. Total number of lung metastases per section is given after pooling both clones of the same condition (CTR, WT, KD). Eighteen mice of each condition CTR, WT and KD were analyzed.
  • CTR empty control vector
  • WT wild-type
  • KD the kinase-dead mutant
  • NME4 is a good prognosis factor in human cancer.
  • A invasive breast carcinoma
  • B ovarian serous cystadenocarcinoma
  • lung carcinoma C
  • D pancreatic ductal adenocarcinoma
  • E uterine corpus endometrial carcinoma
  • E esophageal squamous cell carcinoma
  • G paraganglioma
  • H sarcoma
  • T-RexTM HeLa cells and the pcDNA4/TO vector were obtained from Invitrogen (ThermoFischer Scientific). Constructs to express the NDPK-D WT or NDPK-D mutated at Hisl51 in the catalytic site (Hl 5 IN) or at Arg90 at the cardiolipin binding site (R90D) were obtained as described (7). Recombinant expression and purification of NDPK-D, as well as generation of anti-human NDPK-D polyclonal antibodies in rabbits are described elsewhere (6,). Specific primary antibodies against NDPK-A and B were obtained and used as described in Boissan et al. (11).
  • Mouse monoclonal antibodies anti-Mn-superoxide dismutase (SOD), anti-S100A4, anti-Fascin, anti-alpha-tubulin and anti -tubulin beta II were from Bender Medsystems GmbH (Vienna, Austria), Abnova (Taipei, Taiwan), Agilent (Santa Clara, CA, USA), Sigma-Aldrich and Abeam (Cambridge, MA, USA), respectively. Rabbit monoclonal anti -gamma synuclein was obtained from Abeam.
  • Polyclonal goat anti-ISG15, mouse monoclonal anti-phospho-Thr202Tyr204 ERK1/2 and rabbit polyclonal anti-ERKl/2 and cyclin A were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
  • Rabbit monoclonal anti -phospho-Tyr 1068 EGFR, rabbit monoclonal anti-EGFR, rabbit polyclonal anti-phospho- Ser473 AKT, rabbit polyclonal anti-AKT, rabbit polyclonal anti-phospho-Ser9 GSK3P, mouse monoclonal anti-GSK3p rabbit polyclonal anti-phospho-Ser 199/204 PAK1, and rabbit polyclonal anti-PAKl were from Cell Signaling Technology Inc.
  • Mouse monoclonal anti -RAC 1 was from BD Biosciences (San Jose, CA, USA).
  • Mouse monoclonal anti-a tubulin was from Sigma-Aldrich (St-Louis, MO, USA.
  • Mouse monoclonal anti-Cyclin Bl and PCNA were from Neomarkers (Fremont, CA, USA) and DakoCytomation (Glostrup, Denmark), respectively.
  • Pharmacological inhibitors of PI3K (GSK2126458) and Src (MA475271) were obtained from GlaxoSmithKline (GSK, Brentford, UK) and AstraZeneca (AZ, London, UK), respectively.
  • Pharmacological inhibitors of p38 (SB203580), JNK (SP600125), and EGFR (lapatinib) were purchased from Selleckchem (Houston, TX, USA).
  • Human EGF was purchased from PeproTech (Rocky Hill, NJ, USA).
  • T-RexTM HeLa cells were stably transfected with the vector pcDNATM4/TO without insert (control) or with an insert coding for the NDPK-D WT or the NDPK-D mutants (Hl 5 IN and R90D) as described (7, 8).
  • cells were grown in 3.5 cm diameter Petri dishes or in 6 well plates, were rinsed twice with ice-cold PBS and lysed in 50 pl RIPA/well containing anti-proteases (Calbiochem, cocktail set III or Complete®, Sigma), anti -phosphatases (Sigma, cocktail n°2) and 1 mM EDTA.
  • the lysate was either used immediately or frozen in liquid nitrogen and stored at 20°C until use.
  • citrate synthase activity measurements the fresh lysate was sonicated for additional 5 sec at 50% power and centrifuged at 10,000xg for 20 min at 4 °C and the supernatant kept. Crude HeLa mitochondria were isolated by differential centrifugation according to Eskes et al. (12). The protein concentration was determined by a BCA protein assay (Pierce), using bovine serum albumin (BSA) as standard.
  • BSA bovine serum albumin
  • MDA-MB-231, ZR75-1 and HBL100 cell lines were cultured in DMEM containing 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • BT-474, BT-549, HCC-1428, MDA-MB-468 cells were grown in RPML1640 medium containing 10% FBS and 100 U/mL penicillin and 100 pg/mL streptomycin (P/S).
  • P/S streptomycin
  • HCC-1143, HCC-1187, HCC-1599, HCC-1500, and HCC-1937 cells were grown in RPMI-1640 medium containing 10% FBS, P/S, 1.5 g/L sodium bicarbonate, 10 mM Hepes and 1 mM sodium pyruvate.
  • T47D cells were grown in RPMI-1640 medium containing 10% FBS, P/S and 0.2 U/mL bovine insulin.
  • BT-483 cells were grown in RPMI-1640 medium containing 20% FBS, P/S and 0.01 mg/mL bovine insulin.
  • MCF-10A, MCF-10-2A, and 184B5 cells were grown in DMEM-F12 containing 5% horse serum, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin and 500 ng/mL hydrocortisone.
  • MCF-12A cells were grown in DMEM-F12 containing 5% horse serum, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 1.2 g/L sodium bicarbonate, 0.5 mM sodium pyruvate and 15 mM Hepes.
  • HMEC and hTERT-HMEl cells were grown in Mammary Epithelial Cell Growth Medium BulletKit (Lonza, Basel, Switzerland).
  • Hs578T and MDA-MB- 361 cells were grown in DMEM containing with 10% FBS and P/S.
  • BT-20 and MCF-7 cells were grown in MEM containing 10% FBS, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino-acids and 1 mM sodium pyruvate.
  • MDA-MB-157 and MDA-MB-453 cells were grown in Leibovitz's L-15 medium containing 10% FBS, P/S and 10 mM Hepes.
  • MDA-MB-415 cells were grown in Leibovitz's L-15 medium containing 15% FBS, P/S, 10 mM Hepes, 0.01 mg/mL insulin and 0.01 mg/mL glutathione.
  • CAMA1 cells were grown in Eagle’s MEM containing 10% FBS and P/S.
  • MDA-MB-435S cells were grown in Leibovitz's L-15 medium containing 10% FBS, P/S, and 0.01 mg/mL insulin. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.
  • Proteins from cell extracts were electrophoretically separated on 10% or 12.5% SDS polyacrylamide gels and transferred onto Immobilon P membranes (0.1 pm, Millipore) for 2 h at 22 V in 10 mM CAPS buffer, pH 11, 10% methanol for NDPK-D, as described in (7), or onto nitrocellulose membranes for 90 min at 50 V in 0.025 M Tris-base, 0.192 M glycine, 20% methanol, and 0.02% SDS for the other proteins.
  • the polyclonal anti-NDPK-D was diluted 1/7500, the anti-a tubulin (loading control) 1/5000, and the other primary antibodies 1/500. Blots were revealed with appropriate peroxidase-coupled secondary antibodies and ECL Plus substrate (GE Healthcare).
  • Cellular spatial distribution was characterized and quantified using algorithmic programs of cellular sociology based on the use of three previously described geometrical models, namely Voronoi's partition, Delaunay' graph and minimum spanning tree (MST) as described (13, 14).
  • the aggregation assay was performed as reported (15) by seeding cells on top of a gelified agar medium. Aggregate formation was scored under an inverted microscope at xlO magnification after 24 h incubation at 37 °C. Native type I collagen invasion assays were performed as described earlier (14, 16, 17. Two- and three-dimensional migration assays are described in (13, 14) and (18), respectively.
  • Confluent monolayers of cells were washed with ice-cold PBS and separated from the plates by incubation with PBS free of Ca 2+ and 0.6 U/mL of dispase I (MP Biomedicals, Irvine, CA, USA) for 35 min at 37°C.
  • the dispase solution was removed by centrifugating the cells for 2 min at 400xg and replaced by 200 pL of PBS.
  • the cells were mechanically separated by pipetting up and down five times with a 200 pL pipette.
  • the aggregates were observed by light microscopy using the 10X objective (Echo Rebel Microscope, San Diego, CA, USA). The size of the aggregates was measured using the Fiji software (aggregates ⁇ 200 pm 2 were excluded from the quantification). Wound healing assay.
  • Culture media were collected and concentrated using 10 KDa cut-off ultra-centrifugal filter units (Amicon, Merck-Millipore, Burlington, MA, USA). Protein concentration was determined by the Bradford method, and 200 pg of concentrated supernatant proteins were assayed for proteolytic activity on gelatin-substrate gels. Briefly, samples were mixed with nonreducing loading buffer containing 2.5% SDS, 1% sucrose and separated in 8% acrylamide gels co-polymerized with 1 mg/mL gelatin.
  • Electrophoresis was conducted at 80 V for 2.5 h, then the gels were rinsed twice in 2.5% Triton X-100, and then incubated in 50 mM Tris-HCl pH 7.4 and 5 mM CaCh assay buffer at 37 °C for 24 h. Gels were fixed and stained with 0.25% Coomassie Brilliant Blue G-250 in 10% acetic acid and 30% methanol. Proteolytic activity was detected as clear bands against the background stain of undigested substrate in the gel. Quantification was performed using ImageJ2 software (NIH, Bethesda, MD, USA).
  • NDPK activity in mitochondrial extracts (1-10 pg protein/assay) was measured spectrophotometrically by a coupled pyruvate kinase-lactate dehydrogenase assay using 0.2 mM ATP and 0.2 mM TDP as substrates and adding 100 pM Ap5A to inhibit endogenous adenylate kinase, as described previously (6,7).
  • citrate synthase activity was measured in cell lysates in the presence of 150 mM Tris pH 8, 150 pM 5, 5'-dithiobis-(2 -nitrobenzoic acid) (DTNB), 300 pM acetyl-coenzyme A and 500 pM oxaloacetate. Reduction of DTNB by CS at 37°C was followed spectrophotometrically at 412 nm and CS activity calculated in nkat/mg of total protein.
  • DTNB 5'-dithiobis-(2 -nitrobenzoic acid)
  • Lysates were clarified by centrifugation, and equal amounts of protein lysates were incubated with 20 pL of purified glutathione S-transferase (GST)-CRIB (Cdc42/Rac interactive binding motif) immobilized on glutathione-Sepharose beads (17-0756-01, GE Healthcare,) for Ihour rocking at 4°C.
  • GST glutathione S-transferase
  • Cdc42/Rac interactive binding motif immobilized on glutathione-Sepharose beads (17-0756-01, GE Healthcare,) for Ihour rocking at 4°C.
  • the beads were washed 3 times in lysis buffer and boiled for 10 min in SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 0.002% bromophenol blue, 2% SDS, and 5% P-mercaptoethanol).
  • SDS sample buffer 62.5 mM Tris-HCl pH 6.8, 10% g
  • Mitochondrial membrane potential was determined with about 106 HeLa cells per sample, first incubated for 30 min at 37°C with 50 nM TMRM (tetramethylrhodamine, methyl ester Life Technologies, ThermoFisher Scientific, Waltham, Massachusetts, US), a membrane potential sensitive dye, and 100 nM Mitotracker GreenFM (Life Technologies, ThermoFisher Scientific, Waltham, Massachusetts, US).
  • TMRM tetramethylrhodamine, methyl ester Life Technologies, ThermoFisher Scientific, Waltham, Massachusetts, US
  • Mitotracker GreenFM Life Technologies, ThermoFisher Scientific, Waltham, Massachusetts, US.
  • TMRM fluorescence gated by Mitotracker signal was analyzed by FACS (BD LSR FORTESSA, Becton Dickinson, Le Pont-de-Claix, France). Cells were then incubated with 1 pl 50 mM CCCP for another 5 min at room temperature to entirely depolarize the mitochondria, and again analyzed by FACS. About 20000-50000 events gated on Mitotracker fluorescence were measured, and differences in samples before and after addition of CCCP calculated as readout for mitochondrial membrane potential. Laser excitation was 488nm and 532nm for Mitotracker GreenFM and TMRM, respectively. Fluorescence emission was collected with a 530/30 nm band-pass filter for Mitotracker GreenFM and 585/15 nm band-pass filter for TMRM.
  • TMRE tetramethylrhodamine ethyl ester
  • Oxygen consumption in intact HeLa cells was measured in a thermostatically controlled Clark electrode oxygraph at 37°C (Strathkelvin MS200A system). HeLa cells were detached by trypsin and counted. A cell suspension (100 millions of trypan blue negative cells per ml) was prepared in Roswell Park Memorial Institute (RPMI) medium (ThermoFisher, France). Five million cells were added in the oxygraph chamber containing RPMI medium to a final volume of 500 pl. Oxygen consumption of cells was measured with succinate as substrate before and after the addition of oligomycin (0.06 pg/ml) and FCCP (0.5pM), and results expressed as nmol O2 per minute per mg of cellular protein.
  • RPMI Roswell Park Memorial Institute
  • Calcium retention capacity in digitonin permeabilized cells was determined in trypsin- detached HeLa cells (2 million trypan blue negative cells), permeabilized immediately before use by incubation under stirring for 2 min at 30°C in 250 mM sucrose, 10 mM Tris-MOPS, 1 mM Pi -Tris (pH 7.4) supplemented with 100 pg/ml digitonin. Initially, 0.25 pM Calcium Green-5N (Molecular Probes, Eugene, OR, USA) are added, followed by 5 mM succinate, to a final volume of 1 ml. The calcium retention capacity was measured by sequential addition of 12,5 pM Ca2+ pulses until permeability transition occurs (34).
  • Extramitochondrial Ca2+ was measured fluorimetrically at 30°C using a PTI Quantamaster C61 spectrofluorimeter (excitation: 506 nm; emission: 530 nm) (35). Results are expressed as nmol Ca2+ per 2 millions of trypan blue negative cells.
  • Extracellular acidification rate (ECAR) to estimate glycolytic activity was determined by an Agilent Seahorse XF flux analyzer according to manufacturer’s instructions.
  • CM-H2DCFDA CM-H2DCFDA
  • CM-H2DCFDA 9 pM
  • DMEM DMEM without FBS
  • Quantification was performed with a plate fluorescence reader (Spectrafluor Plus, Tecan-France, Trappes, France) at 520 nm (19).
  • Further markers of oxidative stress were analyzed as described in (20), including protein oxidation by thiols groups (SH) (21) and Ferric Reducing Ability of Plasma (FRAP) by ferric reduction (22).
  • SH protein oxidation by thiols groups
  • FRAP Ferric Reducing Ability of Plasma
  • the lipid hydroperoxides were determined using a lipid hydroperoxide assay kit (Cayman Chemical Co., USA) according to the manufacturer’s instructions.
  • Cell extracts were prepared by cell lysis with the cold mixture methanol/water (70/30, v/v) after removing of cell medium and two washes with cold PBS. Extracts were stored at -80°C before analysis. Analysis of nucleoside mono-, di- and triphosphates in cell extracts was performed on an Ultimate 3000 liquid chromatography system (ThermoFisher ScientificTM, Bremen, Germany) coupled with a Q-Exactive Plus Orbitrap mass spectrometer (ThermoFisher ScientificTM, Bremen, Germany) using a validated method (38).
  • Results were expressed as the ratio of triphosphate/diphosphate which corresponds to area of the nucleoside triphosphate peak/area of the nucleoside diphosphate peak and as the ratio of diphosphate/monophosphate which corresponds to area of the nucleoside diphosphate peak/area of the nucleoside monophosphate peak.
  • HeLa clones were seeded at 5000 cells/well into 96-well plates and proliferation was continuously monitored every hour over a time period between 12 and 36 hours. Data analysis was performed using RTCA 1.2 software supplied with the instrument. Levels of proliferation markers, cyclin A, cyclin Bl and PCNA were analyzed by Western blotting of HeLa clone extracts.
  • the supernatants were collected and proteins were precipitated with a 2-D Clean-Up Kit (GE Healthcare) following the manufacturer's instructions.
  • the pellets were solubilized in 100 pL of UTC buffer (UTCD buffer without DTT) and the protein concentration determined using Quick- Start Bradford Dye Reagent (Bio-Rad).
  • Two-dimensional differential in-gel electrophoresis (2D-DIGE).
  • Three independent samples of two independent clones for each condition (control HeLa-Trex cells transfected with empty vector (CTR1A, B, C; CTR2B, C, D); cells overexpressing the wild-type NDPK-D (WT1 A, B, C; WT2A, C, D), the catalytically inactive (KD1 A, B, D; KD2A, B, C) and the CL- binding-deficient enzyme (BD1A, B, C; BD2A, B, D) were analyzed by 2D-DIGE.
  • MS Mass Spectrometry
  • two distinct semi-preparative 2D-gels were prepared using 400 pg of WT and 400 pg of a mix of BD and KD, respectively, to rehydrate the IPG strips. After electrophoresis, 2D-gels were fixed and stained as described in (40). Gels were scanned using a Typhoon 9400 Trio Variable Mode Imager (GE Healthcare) at 488/520 nm, 100 pm resolution. Spots of interest were excised using the Ettan spot picker (GE Healthcare).
  • the Linear Trap Quadrupole Orbitrap mass spectrometer acquired data throughout the elution process and operated in a data dependent scheme with full MS scans acquired with the Orbitrap, followed by up to 20 LTQ MS/MS CID spectra on the most abundant ions detected in the MS scan. The fragmentation was permitted for precursors with a charge state of 2, 3, 4 and above.
  • the software used to generate mgf (Mascot generic format) files was Proteome discoverer vl.4.0.288.
  • the threshold of Signal to Noise for extraction values is 3.
  • siRNAs targeting NME1 Sil 5’-GGCUGUAGGAAAUCUAGUU - SEQ ID NO:3; Si2 5’-GGAUUCCGCCUUGUUGGUC - SEQ ID NO:4 or targeting NME4 (Sil 5’ -AGCACAAGAUUGGACCAAU - SEQ ID NO:5; Si2 5’ GCAAGAACCCAAGCCCACA - SEQ ID NO:6) synthesized by ThermoFisher Scientific (ThermoFisher Scientific, Waltham, MA, USA) were used.
  • the siRNA control sequence was 5’-GGCUGUAGAAGCUAUAGUU (SEQ ID NO:7).
  • Cells were transfected with control or specific siRNA sequence using the DharmaFECT 4 transfection reagent (Dharmacon, Inc, Lafayette, CO, USA).
  • Quantitative PCR was performed on HeLa stable cell lines using the mix 2X Roche LightCycler (480 SY Green Master Mix- ref 4 887 352 001- Roche Diagnostics, Mannheim, Germany) on a Light Cycler 96 Real Time PCR Roche (Roche Diagnostics). Data from each sample were normalized on the basis of its content in HPRT (hypoxanthine-guanine phosphorribosyl transferase) transcripts. The primers used were: ISG15, S10A4, FSCN1, N-cadherin, SYUG, HPRT, CDH2. Data were collected and analyzed with Roche LightCycler® 96 System Software 3.5.3 (Roche Diagnostics). Data were expressed as a relative amount (2' AACT ) of a control experiment used as a calibrator.
  • Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2) statuses were determined at the protein level by biochemical methods (Dextran-coated charcoal method, enzyme immunoassay or immunohistochemistry) and confirmed by real-time quantitative RT-PCR.
  • TBP encoding the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) was selected as an endogenous control due to the moderate level of its transcripts and the absence of known TBP retro-pseudogenes (retro-pseudogenes lead to coamplification of contaminating genomic DNA and thus interfere with RT-PCR transcripts, despite the use of primers in separate exons).
  • NDPK-D mutations induce a morphotypic switch linked to a loss of intercellular adhesion
  • the HeLa clones that are analyzed here in detail have been used already in our earlier studies (7, 8, 10).
  • the control HeLa clones contained empty vector (control, abbreviated as CTR) and expressed low levels of endogenous NDPK-D (data not shown).
  • Clones stably transfected with vectors for different NDPK-D variants namely wild-type (WT), CL-binding deficient (R90D mutation or BD) or kinase-dead (Hl 5 IN mutation or KD), expressed high levels of these NDPK-D proteins, presenting as a single strong band at the size of mature enzyme (data not shown).
  • N-cadherin was markedly decreased in both mutant NDPK-D expressing clones as compared to control and WT NDPK-D expressing cells, again most pronounced for the kinase-dead Hl 5 IN mutant (data not shown). All these data consistently show a clear and similar morphotypic switch that occurred in the mutant NDPK-D expressing clones, with a marked loss of cell-cell aggregation and cell-cell adhesion.
  • NDPK-D mutations increase 2D and 3D cell migration
  • the increased 2D migration speed exhibited by the mutant HeLa clones is also obvious through examination of video microscopy images (data not shown).
  • the 3D migration assay revealed higher migration speed along the x-y-z planes for the two mutant expressing clones (KD1, KD2) as compared to the WT NDPK-D expressing clone (data not shown). Since cell migration is largely mediated by Rho-GTPases, we evaluated Rael activation in these Hela clones via GST-pulldown assays (data not shown).
  • NDPK-D mutations increase the 3D invasive potential
  • NDPK-D mutants affects the ability of HeLa cells to invade a three-dimensional matrix of native type I collagen during 24 hours (data not shown). HeLa cells are notoriously poor in degrading the extracellular matrix (25). When seeded on native type I collagen, mutant NDPK-D formed numerous cellular protrusions, which invaded the collagen layer, while controls and WT enzyme expressing cells presented only few of these (data not shown). Expression of both NDPK-D mutants strongly increased invasion through native type I collagen as compared to WT NDPK-D; the latter was even significantly lower as compared to the control (data not shown).
  • mutant NDPK-D expression was further confirmed by a 14-day invasion assay.
  • sections of the collagen layer were examined two-weeks after seeding the HeLa clones. While the WT clones remained on the surface, the kinase-dead clones deeply penetrated into the collagen layer (data not shown).
  • the invasive program of mutant clones was not related to an advantage of proliferation since their proliferation rates were lower than the one of the wild-type clones, also confirmed by protein levels of proliferation markers such as cyclin A, cyclin Bl, and PCNA that were higher in WT clones than in CTR, BD and KD clones (data not shown).
  • the cellular proteome reveals changes in metastasis-related and mitochondrial proteins
  • the morphotypic switch and the scattered/migratory/invasive phenotype observed for Hela cells expressing NDPK-D mutants are striking features, considering that they are triggered by a single point mutation in a mitochondrial protein. This implies communication between molecular NDPK-D structure/function and cellular behavior in respect to cell adhesion, motility and invasive potential. Since this should be mediated by changes in the cellular proteome, we next performed a comparative 2D-DIGE proteomic study with two independent clones of every experimental group (CTR, WT, KD and BD NDPK-D).
  • IPA Mitochondrial Dysfunction and Oxidative Stress (data not shown). Indeed, among proteins differentially expressed in mutant KD and BD clones vs. WT were many mitochondrial proteins. A marked change was downregulation of several core subunits of ATP synthase: alpha (ATPA: -1.5, -1.7), beta (ATPB: -2.0, -1.9) and delta (ATP5H: -1.4, -1.6), while few changes were detected in the respiratory chain.
  • alpha ATPA: -1.5, -1.7
  • beta ATPB
  • delta ATP5H
  • adenylate kinase 3 adenylate kinase 3
  • GTP AMP phosphotransferase
  • IMMT MICOS complex subunit MIC60
  • VDACs voltage-dependent anion channels
  • NDPK-D mutations affect mitochondrial structure and function
  • NDPK-D loss-of-function has primary effects on mitochondria.
  • MnSOD mitochondrial protein Mn-superoxide dismutase
  • Both NDPK-D mutant clones showed fragmentation of the network as compared to WT and control cells, determined by decreased filament length, elongation, surface area, and interconnectivity (data not shown).
  • the WT clone had higher elongation and surface area parameters as compared to controls.
  • NDPK-D mutants led to mitochondrial fragmentation, consistent with the key role of NDPK-D in fueling the mitochondrial fusion protein OPA1 (9). Similar networks were observed with MitoTracker Green live stained cells (not shown). Correlated with fragmentation, NDPK-D mutant clones also had lower mitochondrial mass as compared to WT and control cells, consistent with a preferential elimination of fragmented, smaller mitochondria (data not shown).
  • the energy sensor AMP-activated protein kinase (AMPK) was phosphorylated and activated in BD and KD clones relative to WT, also observed with phosphorylation of the AMPK substrate acetyl-CoA carboxylase in BD clones (data not shown).
  • the mitochondrial isoform of creatine kinase (umtCK) was upregulated in both BD and KD clones relative to WT, and the mitochondrial adenylate kinase AK2 was upregulated in the BD clone only (data not shown). Upregulation of these kinases in the mitochondrial intermembrane space often occurs as a compensatory response under energy stress (31).
  • ROS reactive oxygen species
  • NDPK-D is a gatekeeper against EMT in breast cancer cells
  • MDA-MB-231 and ZR75-1 For genetic manipulation of functional NDPK-D levels in breast cancer, we chose two of these human breast tumor cell lines, MDA-MB-231 and ZR75-1. The former had the lowest level of NME4 mRNA and was highly invasive and metastatic, while the latter had the highest level of NME4 mRNA and was minimally invasive with an epithelial-like phenotype (data not shown).
  • the control MDA-MB-231 clones containing empty vector (CTR) expressed undetectable levels of endogenous NDPK-D (data not shown).
  • MDA-MB-231 clones exhibited an immunostaining with anti-NDPK-D antibodies strictly colocalizing with mitochondria- selective marker (data not shown).
  • ZR75-1 cells we did the contrary experiment, depleting NDPK-D specifically by expressing two different siRNAs.
  • Western blotting confirmed the effective siRNA-mediated knockdown of NDPK-D (data not shown).
  • Migrating cells achieve this process by secreting Zn 2+ -dependent MMPs that respond to growth factors, cytokines and hormones (48,49).
  • the MDA-MB-231 clones overexpressing WT NDPK-D as compared to control clones showed a decrease by 60% and 80% in the gelatinase activity of MMP9 and MMP2, respectively (Figure 1C), consistent with their impaired wound healing.
  • NDPK-D depletion increased the secretion of MMP9 by 1.5-fold (data not shown), consistent with accelerated wound healing in this case. MMP2 activation was undetectable in these cells.
  • results obtained with breast cancer MDA-MB-231 and ZR75-1 cells are consistent with our data on cervical cancer HeLa cells, namely showing increased cell motility, reduced cell-cell adhesion, and mitochondrial dysfunction with NDPK-D downregulation or loss-of-function mutations. This strongly supports our conclusion that NDPK-D expression is negatively associated with breast cancer progression and invasion.
  • NME4 expression is negatively associated with EMT and tumor invasion markers and is associated with beneficial clinical outcome in human cancer
  • NDPK-D Based on our novel findings on anti-invasive and anti-metastatic functions of NDPK-D, we predicted that its expression might be down-regulated in human aggressive tumors showing EMT and invasion in comparison to tumors with a good prognosis.
  • NME4 mRNA levels of NME4, CDH1 and KRT18, the genes encoding NDPK-D and two well-known epithelial markers E-cadherin and cytokeratin 18, respectively, in an important cohort of 526 human breast tumors from patients with well-documented follow-up by using RT-qPCR (Table 1). Consistently, we found a strong positive association of NME4 with CDH1 and KRT18 (data not shown).
  • NME4 mRNA levels in this cohort were the lowest in the most aggressive human breast tumors with worst prognosis, the so-called triple-negative breast tumors (data not shown). Similar associations were observed with another metastasis suppressor in this cohort : NME1 (data not shown).
  • NME1 Using the same cohort, a positive association was observed between NME1 and CDH1 and KRT18 and a negative one with the mesenchymal marker VIM, the vimentin gene (data not shown). In addition, NME1, like NME4, is rarely mutated (data not shown).
  • NME4 expression was associated with a poor prognosis ( Figure 4A,B,C,D,E,F); this is also the case for tumors other than carcinomas such as pheochromocytoma, paraganglioma and sarcoma ( Figure 4G,H).
  • Boissan M et al. (2009) The mammalian Nm23/NDPK family: from metastasis control to cilia movement. Mol Cell Biochem 329(l-2):51-62.
  • nucleoside diphosphate kinase D (NM23-H4) binds the inner mitochondrial membrane with high affinity to cardiolipin and couples nucleotide transfer with respiration. J Biol Chem 283(38):26198-26207.
  • Boterberg T Vennekens KM, Thienpont M, Mareel MM, & Bracke ME (2000) Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16.
  • Faure P Lafond JL: Measurement of plasma sulfhydryl and carbonyl groups as a possible indicator of protein oxidation.
  • Favier A.E. Cadet J., Kalyanaraman B., Fontecave M., JL. P. Basel: Birkhaiiser; 1995: 237-248.
  • Shevchenko A Shevchenko A. Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal Biochem. 2001;296(2):279-83.

Abstract

The present invention relates to the prediction and treatment of cancers. In this study, the inventors analyzed how invalidation of the mitochondrial nucleoside diphosphate kinase (NDPK-D) functions affects the cellular behavior. HeLa cells, which naturally express low levels of NDPK-D, were stably transfected with expression vectors, either empty or designed to express NDPK-D wild type or mutant proteins. Single point mutations were chosen to suppress either the catalytic NDPK activity or its ability to bind the mitochondria inner membrane. Both loss-of-function mutations promoted altered mitochondrial structure and function, epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. In human cancer, NME4 expression is negatively associated with tumor aggressiveness, and is a good prognosis factor for beneficial clinical outcome. Thus, the invention relates to a method for predicting the survival time of a patient suffering from a cancer comprising determining the expression level of NDPK-D and to the treatment of cancer in a subject in need thereof by using the NDPK-D protein or a fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.

Description

USE OF NPDK-D TO EVALUATE CANCER PROGNOSIS
FIELD OF THE INVENTION:
The invention relates to a method for predicting the survival time of a patient suffering from a cancer comprising determining the expression level of NDPK-D and to the treatment of cancer in a subject in need thereof by using the NDPK-D protein or a fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.
BACKGROUND OF THE INVENTION:
Carcinomas, the most prevalent malignancies in humans, arise from normal epithelial tissues in a multistep progression from benign precursor lesions. Metastasis, the final step in malignancy, is the cause of death for more than 90% of cancer patients. Molecular mechanisms underlying metastasis have to be elucidated for accurate detection and treatment (1). During metastatic disease, complex pathways involving the tumor cell and the microenvironment mediate tumor invasion at the primary site, survival and arrest in the bloodstream, extravasation, and colonization at a secondary site. The first step in the metastatic cascade, i.e. the breakdown of epithelial intercellular adhesion and the acquisition of an invasive program, allows epithelial cancer cells to breach the basement membrane and to invade stromal type I fibrillar collagen. These events are referred as epithelial-mesenchymal transition (EMT) and are considered crucial events in malignancy yet poorly understood (2). During EMT, epithelial cells lose some of their epithelial characteristics, including cell adhesion and cell polarity; cytoskeletal rearrangement occurs that ultimately leads to an increased motility and an invasive phenotype.
The first metastasis suppressor gene discovered, NME1/NM23-H1 (3) encodes the nucleoside diphosphate kinase A (NDPK-A), which catalyzes synthesis of nucleoside triphosphates including GTP from corresponding nucleoside diphosphates and ATP. In human, ten isoforms of the NME/NM23/NDPK family have been identified (reviewed in (4)), among those, the mitochondrial isoform NME4/NM23-H4, also called NDPK-D (5, 6). If NME1 has a well-known anti-metastatic activity, the contribution of the other isoforms including NME2 and NME3 is much less documented or even unknown for NME4.
NME4/NM23-H4, further only called NDPK-D, is a mitochondrial enzyme, which binds to the mitochondrial inner membrane (MIM) through anionic phospholipids, mainly cardiolipin (CL), and is principally oriented towards the intermembrane space (5-7). The enzyme acts as a lipid-dependent mitochondrial switch with dual function: (i) in phosphotransfer serving, in particular, for local GTP supply to the mitochondrial dynamin GTPase, Optic Atrophy 1 (OPA1), a driver of mitochondrial fusion (8, 9) and (ii) in CL transfer from the inner to the outer membrane, where this phospholipid serves as pro-mitophagic or pro- apoptotic signal (8, 10).
SUMMARY OF THE INVENTION:
In this study, the inventors analyzed how invalidation of NDPK-D functions affects the cellular behavior. HeLa cells, which naturally express low levels of NDPK-D, were stably transfected with expression vectors, either empty or designed to express NDPK-D wild type or mutant proteins. Single point mutations were chosen to suppress either the catalytic NDPK activity of the enzyme or its ability to bind CL, which localizes the enzyme to the inner membrane and is essential for its function in CL intermembrane transfer. Both mutations led to similar alterations in the cellular behavior, linked to altered mitochondrial structure and function, reprogramming of protein expression, and a morphotypic switch towards a pro- metastatic phenotype. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. In human cancer, NME4 expression is negatively associated with tumor aggressiveness, and is a good prognosis factor for beneficial clinical outcome.
Thus, the invention relates to a method for predicting the survival time of a patient suffering from a cancer comprising determining the expression level of NDPK-D and to the treatment of cancer in a subject in need thereof by using the NDPK-D protein or a fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression. Particularly, the invention is defined by its claims.
DETAILED DESCRIPTION OF THE INVENTION:
Diagnostic applications
A first aspect of the invention relates to a method for predicting the survival time of a patient suffering from a cancer, comprising i) determining the expression level of NDPK-D in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is higher than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is lower than its predetermined reference value. Another aspect of the invention relates to a method for predicting the invasiveness of a cancer, comprising i) determining in a sample obtained from the patient the expression level of NDPK-D ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is higher than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is lower than its predetermined reference value.
According to the invention, the cancer may be a solid or a liquid cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a solid metastatic cancer.
According to the invention, the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, uterine cancer, pheochromocytoma, paraganglioma and sarcoma.
According to these particular embodiments, the cancer may be selected in the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, uterine cancer, esophageal cancer, pheochromocytoma, paraganglioma and sarcoma.
In particular, the cancer may be selected in the group consisting of breast carcinoma, ovarian serous cystadenocarcinoma, lung carcinoma, pancreatic ductal adenocarcinoma, uterine corpus endometrial carcinoma, esophageal squamous cell carcinoma, pheochromocytoma, paraganglioma and sarcoma.
According to these particular embodiments, the sample can be blood, peripheral -blood, serum, plasma, tumoral circulating cells, tumor sample that is to say a sample obtained from the tumor or a biopsy obtained from the tumor or mitochondria purified from the tumor. In some embodiments, the sample is a solid tumor sample. By way of comparison, the sample can also arise from the uninvolved corresponding tissue sample. As used herein, the term “subject” or “patient” denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with a cancer, particularly a metastatic cancer.
As used herein, the term “NDPK-D” for “nucleoside diphosphate kinase-D”, also known as NME4/NM23-H4, is a ubiquitous mitochondrial enzyme that catalyzes transfer of gammaphosphates, via a phosphohistidine intermediate, between nucleoside and deoxynucleoside tri- and diphosphates. For the gene sequence, its Entrez Gene reference number is: 4833; for the protein, the UniProt reference number is: 000746 and NCBI reference is: NP 005000.1; for the mRNA, the GenBank reference is: Y07604.1.
As used herein, the term “survival time” denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as pancreatic cancer (according to the invention). The survival time rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.
As used herein and according to the invention, the term “survival time” can regroup the term “Overall survival (OS)”.
As used herein, the term OS denotes the time from diagnosis of a disease such as cancer (according to the invention) until death from any cause. The overall survival rate is often stated as a two-year survival rate, which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.
As used herein, the term “invasiveness of a cancer” is the capacity of a cancer to lead to the formation of metastases.
Measuring the expression level of NDPK-D can be done by measuring the gene expression level of NDPK-D or by measuring the level of the protein NDPK-D and can be performed by a variety of techniques well known in the art.
Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR). Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence or mRNA) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrromethene boron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).
In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOT® (obtained, for example, from Life Technologies (Quantum Dot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the band gap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif.).
Additional labels include, for example, radioisotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+. Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).
Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pinkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. l. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.
Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following nonlimiting examples: biotin, digoxigenin, DNP (dinitrophenol), and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acid sequences (e.g., target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can be labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 nm). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can be envisioned, all of which are suitable in the context of the disclosed probes and assays.
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources. According to the invention, the level of NDPK-D proteins may also be measured and can be performed by a variety of techniques well known in the art. For measuring the expression level of NDPK-D, techniques like ELISA (see below) allowing to measure the level of the soluble proteins are particularly suitable. In a particular embodiment, the sample is solubilized since NDPK-D is bound to the mitochondrial membrane.
In the present application, the “level of protein” or the “protein level expression” or the “protein concentration” means the quantity or concentration of said protein. In another embodiment, the “level of protein” means the level of NDPK-D protein fragments. In still another embodiment, the “level of protein” means the quantitative measurement of NDPK-D protein expression relative to a negative control.
In a particular embodiment, for example, the proteins in the sample or mitochondria isolated thereof are solubilized with detergent, since NDPK-D is bound to the mitochondrial membrane. The proportion of NDPK-D that remains in the particulate fraction without detergent treatment and that is above a predetermined reference value would relate to membrane-bound protein necessary for metastasis-suppression function. It would therefore have predictive value. In another particular embodiment, enzymatic activity of NDPK-D is measured in mitochondria isolated form tumour tissue.
Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresismass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
Methods of the invention may comprise a step consisting in comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the protein NDPK-D. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the protein NDPK-D. Thus, in a particular embodiment, fragments of NDPK-D protein may also be measured.
Predetermined reference values used for comparison of the expression levels may comprise “cut-off’ or “threshold” values that may be determined as described herein. Each reference (“cut-off’) value for NDPK-D level may be predetermined by carrying out a method comprising the steps of: a) providing a collection of samples from patients suffering of a cancer and/or samples of the corresponding uninvolved tissues as described in the invention; b) determining the level of NDPK-D for each sample contained in the collection provided at step a); c) ranking the tumor tissue samples according to said level d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level, e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient; f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve; g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets h) selecting as reference value for the level, the value of level for which the p value is the smallest.
For example the expression level of NDPK-D has been assessed for 100 cancer samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the highest expression level and sample 100 has the lowest expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.
In routine work, the reference value (cut-off value) may be used in the present method to discriminate cancer samples and therefore the corresponding patients.
Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.
The man skilled in the art also understands that the same technique of assessment of the expression level of a protein should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a protein of a patient subjected to the method of the invention
A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of NDPK-D in the sample obtained from the patient.
The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively the kit of the invention may comprise amplification primers that may be prelabelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
The present invention also relates to NDPK-D as biomarkers for outcome of cancer patients.
The present invention also relates to NDPK-D as biomarkers of invasiveness of cancer.
Therapeutic applications
In a second aspect, the invention relates to the NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression for use in the treatment of cancer in a subject in need thereof.
In a particular embodiment the NDPK-D protein or fragment thereof or a fusion protein thereof or the agent for NDPK-D protein expression will be useful to treat metastasis.
The invention also relates to a method for treating a cancer in a subject in need thereof comprising the administration to said subject the NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.
As used herein, the term “an agent for NDPK-D protein expression” denotes an agent which can increase or restore the NDPK-D protein expression or increase the activity of the NDPK-D. To increase or restore the NDPK-D protein expression, the agent can also increase or restore the NDPK-D gene expression. In some embodiments, the agent for NDPK-D protein expression is a vector encoding NME4, NME4 mRNA or is the NME4 mRNA molecule itself. The NME4 mRNA molecule may be included in an appropriate vector. As example, the appropriate vector may be a membrane or lipid vesicle.
In order to test the functionality of a putative agent for NDPK-D protein expression a test is necessary. For that purpose, to identify agent for NDPK-D protein expression, tools like anti-NDPK-D antibody can be used to detect variation (and thus an increase) in the level of the NDPK-D protein. ELISA test or western blotting for example can be used to detect an increasing of the level of NDKP-D after use of an agent for NDPK-D protein expression. According to the invention, the cancer may be a solid or a liquid cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a solid metastatic cancer.
According to the invention, the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, uterine cancer, pheochromocytoma, paraganglioma and sarcoma.
According to these particular embodiments, the cancer may be selected in the group consisting of breast cancer, ovarian cancer, lung cancer, pancreatic cancer, uterine cancer, esophageal cancer, pheochromocytoma, paraganglioma and sarcoma.
In particular, the cancer may be selected in the group consisting of breast carcinoma, ovarian serous cystadenocarcinoma, lung carcinoma, pancreatic ductal adenocarcinoma, uterine corpus endometrial carcinoma, esophageal squamous cell carcinoma, pheochromocytoma, paraganglioma and sarcoma.
As used herein and according to the invention, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder (like cancer) or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
In some embodiments, the NDPK-D protein of the invention is an isolated, synthetic or recombinant NDPK-D protein.
In some embodiments, said NDPK-D protein comprises a sequence as set forth by SEQ ID NO: 1:
MGGLFWRSAL RGLRCGPRAP GPSLLVRHGS GGPSWTRERT LVAVKPDGVQ RRLVGDVIQR FERRGFTLVG MKMLQAPESV LAEHYQDLRR KPFYPALIRY MSSGPVVAMV WEGYNVVRAS RAMIGHTDSA EAAPGTIRGD FSVHISRNVI HASDSVEGAQ REIQLWFQSS ELVSWADGGQ HSSIHPA
In some embodiments, the protein of the present invention comprises or consists of an amino acid sequence having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % of identity with the SEQ ID NO: 1.
According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100% of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). In particular the polypeptide of the invention is a functional conservative variant of the polypeptide according to the invention. As used herein the term “function-conservative variant" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "function-conservative variant" also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent polypeptide to which it is compared.
A further aspect of the present invention relates to a fusion protein comprising the protein according to the invention that is fused to at least one heterologous polypeptide.
The invention relates also to a fusion protein which consist to the protein according to the invention fused to at least one heterologous polypeptide.
The term “fusion protein” refers to the protein or peptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.
According to the invention, the fusion protein comprises the protein or peptide that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.
As used herein, the term “directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the protein or peptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.
In other words, in this embodiment, the last amino acid of the C-terminal end of said protein or peptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said protein or peptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.
As used herein, the term “spacer” refers to a sequence of at least one amino acid that links the protein or peptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances.
In some embodiments, the heterologous polypeptide is a cell -penetrating peptide, a Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide or a membranous penetrating sequence.
The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT, mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The proteins, peptides or fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides or fusion proteins, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, the polypeptides or fusion proteins of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well- known techniques.
The proteins, peptides, fusion proteins or the NME4 mRNA molecule itself of the invention can be used in an isolated (e.g., purified) form or contained in a vehicle, such as a membrane or lipid vesicle (e.g. a liposome).
In specific embodiments, it is contemplated that proteins, polypeptides or fusion proteins according to the invention may be modified in order to improve their therapeutic efficacy and their stability using well-known techniques. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
A strategy for improving drug stability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
For example, PEGylation, i.e. covalent attachment of one or more polyethyleneglycol (PEG) groups, is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002). The benefits include among others: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles et.al., 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide.
Therefore, advantageously, the proteins, peptides or fusion proteins of the invention may be covalently linked with one or more PEG group(s). One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc.
In one embodiment, the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH ("methoxy PEG"). In addition, such a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythritol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., 1995).
To effect covalent attachment of PEG groups to the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N- hydroxysuccinimide (NHS), aldehyde, nitrophenyl carb onate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e. g. from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK. Alternatively, the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90/13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs : NHS-PEG (e g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.
The conjugation of the proteins, peptides or fusion proteins and the activated polymer molecules is conducted by use of any conventional method. Conventional methods are known to the skilled artisan. The skilled person will be aware that the activation method and/or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).
In one embodiment, polypeptides are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH2), C (for SH if at least one cysteine is conserved) or/and Q and N (for the amide function).
In one embodiment, additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In another embodiment, additional PEGylation sites are chemically introduced by modifying amino acids on polypeptides of the invention.
In one embodiment, PEGs are conjugated to the polypeptides or fusion proteins through a linker. Suitable linkers are well known to the skilled person. A preferred example is cyanuric chloride ((Abuchowski et al., 1977); US 4,179, 337).
Conventional separation and purification techniques known in the art can be used to purify pegylated polypeptides of the invention, such as size exclusion (e.g. gel filtration) and ion exchange chromatography. Products may also be separated using SDS-PAGE.
In one embodiment, the pegylated polypeptides provided by the invention have a serum half-life in vivo of at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified polypeptide.
In some embodiments, the agent for NDPK-D protein expression of the invention is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for NDPK-D protein, a nucleic acid sequence encoding for the fusion protein, a nucleic acid encoding a fragment of a NDPK-D protein, a cell expressing NDPK-D protein, and agent inducing NDPK-D gene expression and their combinations.
In some embodiments, said nucleic acid encoding for NDPK-D protein comprises a sequence as set forth by SEQ ID NO: 2.
SEQ ID NO: 2: ggccgggcgt catgggcggc ctcttctggc gctccgcgct gcgggggctg cgctgcggcc cgcgggcccc gggcccgagc ctgctagtgc gccacggctc gggagggccc tcctggaccc gggagcggac cctggtggcg gtgaagcccg atggcgtgca acggcggctc gttggggacg tgatccagcg ctttgagagg cggggcttca cgctggtggg gatgaagatg ctgcaggcac cagagagcgt ccttgccgag cactaccagg acctgcggag gaagcccttc taccctgccc tcatccgcta catgagctct gggcctgtgg tggccatggt ctgggaaggg tacaatgtcg tccgcgcctc aagggccatg attggacaca ccgactcggc tgaggctgcc ccaggaacca taaggggtga cttcagcgtc cacatcagca ggaatgtcat ccacgccagc gactccgtgg agggggccca gcgggagatc cagctgtggt tccagagcag tgagctggtg agctgggcag acgggggcca gcacagcagc atccacccag cctgaggctc aagctgccct taccacccca tcccccacgc aggaccaact acctccgtca gcaagaaccc aagcccacat ccaaacctgc ctgtcccaaa ccacttactt ccctgttcac ctctgcccca ccccagccca gaggagtttg agccaccaac ttcagtgcct ttctgtaccc caagccagca caagattgga ccaatccttt ttgcaccaaa gtgccggaca acctttgtgg tggggggggg tcttcacatt atcataacct ctcctctaaa ggggaggcat taaaattcac tgtgcccag
In some embodiments, the nucleic acid encoding for NDPK-D protein for example comprises or consists of a sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % identical to sequence SEQ ID NO: 2.
As used herein, a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
These nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art. Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
So, a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a polypeptide or a fusion protein of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors.
As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
Any expression vector for animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al., 1984), pKCR (O'Hare et al., 1981), pSGl beta d2-4 (Miyaji et al., 1990) and the like.
Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral, lentiviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, Psi CRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.
Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana et al., 1987), promoter (Mason et al., 1985) and enhancer (Gillies et al., 1983) of immunoglobulin H chain and the like.
A further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a protein or a fusion protein according to the invention or a vector according to the invention. In particular, a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention.
The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".
In a particular embodiment, for expressing and producing proteins, peptides or fusion proteins of the invention, prokaryotic cells, in particular E. coli cells, will be chosen. Actually, according to the invention, it is not mandatory to produce the polypeptide or the fusion protein of the invention in a eukaryotic context that will favour post-translational modifications (e.g. glycosylation). Furthermore, prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21, COS-7, C127, PER.C6, YB2/0, HEK293, mononuclear macrophage/monocyte-lineage hematopoietic precursors, Haematopoietic stem cells, Mononuclear precursor cells, osteoblast or inactive osteoclast could be used, for their ability to process to the right post-translational modifications of the fusion protein of the invention. The construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques. The protein, peptide or the fusion protein of the invention, can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the polypeptide or the fusion protein expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.
A further aspect of the invention relates to a method for producing a protein, peptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said protein, peptide or fusion protein; and (ii) recovering the expressed protein, peptide or fusion protein.
In some embodiments, the agent for NDPK-D protein expression of the invention is an agent inducing NDPK-D gene and peptide expression selected from the group consisting of, but not limited to, Human Cytomegalovirus (HCMV), VHL/E HCMV strain, and TB40/E HCMV strain.
In a third aspect, the invention relates to an anti-cancer agent for use in the treatment of a cancer in a patient with a bad prognosis as described above.
In a particular embodiment, the invention also relates to the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention for use in the treatment of a cancer in a patient with a bad prognosis as described above.
In a particular embodiment, the invention relates to an anti-cancer agent in combination with the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention for use in the treatment of a cancer in a patient with a bad prognosis as described above.
The invention also relates to a method for treating a cancer in a patient with a bad prognosis as described above comprising the administration to said patient of an anti-cancer agent.
The invention also relates to a method for treating a cancer in a patient with a bad prognosis as described above comprising the administration to said patient of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention.
Anti-cancer agent can be selected in the group consisting in cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
Additional anti-cancer agent may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
In the present methods for treating cancer, additional therapeutic active agent can be added like an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.
In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
In still another embodiment, the other therapeutic active agent can be an opioid or nonopioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.
In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies. The invention also relates to a pharmaceutical composition comprising A NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression for use in the treatment of a cancer in subject in need thereof.
Another aspect of the invention relates to a pharmaceutical composition comprising an anti-cancer treatment for use in the treatment of cancer in a subject with a bad prognosis as described above.
Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.
Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
Typically the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention as described above or the anti -cancer agent according to the invention are administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention as above described or the anti-cancer agent is meant a sufficient amount of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent for treating cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or anti-cancer agent will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anticancer agent employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent employed; the duration of the treatment; drugs used in combination or coincidental with the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or anti-cancer agent at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent, preferably from 1 mg to about 100 mg of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
In a particular embodiment, the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression according to the invention or the anti-cancer agent may be used in a concentration between 0.01 pM and 20 pM, particularly, the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the invention or the anti-cancer agent may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM.
According to the invention, the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent is administered to the subject in the form of a pharmaceutical composition. Thus, the invention also relates to a therapeutic composition comprising the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression or an anti-cancer agent for use in the treatment of a cancer in a subject in need thereof.
Typically, the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the invention or the anti-cancer agent of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti-cancer agent of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the NDPK-D protein or fragment thereof and/or an agent for NDPK-D protein expression of the present invention or the anti -cancer agent of the invention plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: Migration, invasion, and MMP activity of MDA-MB-231 cells genetically modified for NDPK-D and of ZR75-1 cells depleted for NDPK-D. (A) Quantification of the wound healing assay in ZR75-1 cells. Two different siRNA targeting NME4 were used. Time zero represents confluent monolayer wounds at Oh. Wounds were monitored for 120h after performing the scratch in which knockdown monolayers became fully closed. Data show means ± SEM (n=3). *****p<0.00001 relative to scramble control (Scr). (B) Type I collagen invasion assay of MDA-MB-231 cells. Two MDA-MB-231 clones for each conditions were analyzed named after expressed NDPK-D: CTR, control/empty vector; WT, wild-type; BD, CL-binding- deficient mutant; KD, kinase-dead mutant. Data show means ± SEM. ***p<0.001 relative to control/empty vector (CTR). (C). Quantification of the MMP activity of MDA-MB-231 clones described in B. Bar graphs represent the densitometric and statistical analyses of the bands obtained by gelatin zymography for MMP9 (left) and MMP2 (right) of four independent biological replicates. Concentrated culture media from MCF7 cells was used as positive control. Data show means ± SEM (n=4). *****p<0.00001 relative to control/empty vector (CTR).
Figure 2: Adhesion properties of MDA-MB-231 cells genetically modified for NDPK-D and of ZR75-1 cells depleted for NDPK-D. (A) Two MDA-MB-231 clones were analyzed for each conditions named as described in the legend to Figure 1. The size of the aggregates observed is depicted as the area of their horizontal projections. Data show means ± SEM of three independent biological replicates imaged. *****p<0.00001 relative to control/empty vector (CTR). (B) Two different siRNA targeting NME4 were used for ZR75-1 cell aggregation assay. The size of the aggregates observed in light microscopy representative images is depicted as the area of their horizontal projections. Data show means ± SEM of three independent biological replicates imaged. *****p<0.00001 relative to scramble control (Scr).
Figure 3: NME4-related metastasis-suppression. Experimental metastasis assay, where two HeLa clones for each condition, empty control vector (CTR), wild-type (WT), and the kinase-dead mutant (KD), were injected in the tail vein of nude mice. After 13 weeks, mice were sacrificed, lungs removed, and the number of lung metastases counted. Total number of lung metastases per section is given after pooling both clones of the same condition (CTR, WT, KD). Eighteen mice of each condition CTR, WT and KD were analyzed.
Figure 4: NME4 is a good prognosis factor in human cancer. Kaplan-Meier analysis of overall survival according to NME4 mRNA expression in KM plotter database of invasive breast carcinoma (A), ovarian serous cystadenocarcinoma (B), lung carcinoma (C), pancreatic ductal adenocarcinoma (D), uterine corpus endometrial carcinoma (E), esophageal squamous cell carcinoma (F), pheochromocytoma and paraganglioma (G), and sarcoma (H).
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Table 1: Characteristics of the 526 breast tumors
EXAMPLE:
Material & Methods
Materials
T-RexTM HeLa cells and the pcDNA4/TO vector were obtained from Invitrogen (ThermoFischer Scientific). Constructs to express the NDPK-D WT or NDPK-D mutated at Hisl51 in the catalytic site (Hl 5 IN) or at Arg90 at the cardiolipin binding site (R90D) were obtained as described (7). Recombinant expression and purification of NDPK-D, as well as generation of anti-human NDPK-D polyclonal antibodies in rabbits are described elsewhere (6,). Specific primary antibodies against NDPK-A and B were obtained and used as described in Boissan et al. (11). Mouse monoclonal antibodies anti-Mn-superoxide dismutase (SOD), anti-S100A4, anti-Fascin, anti-alpha-tubulin and anti -tubulin beta II were from Bender Medsystems GmbH (Vienna, Austria), Abnova (Taipei, Taiwan), Agilent (Santa Clara, CA, USA), Sigma-Aldrich and Abeam (Cambridge, MA, USA), respectively. Rabbit monoclonal anti -gamma synuclein was obtained from Abeam. Polyclonal goat anti-ISG15, mouse monoclonal anti-phospho-Thr202Tyr204 ERK1/2 and rabbit polyclonal anti-ERKl/2 and cyclin A were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Rabbit monoclonal anti -phospho-Tyr 1068 EGFR, rabbit monoclonal anti-EGFR, rabbit polyclonal anti-phospho- Ser473 AKT, rabbit polyclonal anti-AKT, rabbit polyclonal anti-phospho-Ser9 GSK3P, mouse monoclonal anti-GSK3p rabbit polyclonal anti-phospho-Ser 199/204 PAK1, and rabbit polyclonal anti-PAKl were from Cell Signaling Technology Inc. (Beverly, MA, USA). Mouse monoclonal anti -RAC 1 was from BD Biosciences (San Jose, CA, USA). Mouse monoclonal anti-a tubulin was from Sigma-Aldrich (St-Louis, MO, USA. Mouse monoclonal anti-Cyclin Bl and PCNA were from Neomarkers (Fremont, CA, USA) and DakoCytomation (Glostrup, Denmark), respectively.
Pharmacological inhibitors of PI3K (GSK2126458) and Src (MA475271) were obtained from GlaxoSmithKline (GSK, Brentford, UK) and AstraZeneca (AZ, London, UK), respectively. Pharmacological inhibitors of p38 (SB203580), JNK (SP600125), and EGFR (lapatinib) were purchased from Selleckchem (Houston, TX, USA). Human EGF was purchased from PeproTech (Rocky Hill, NJ, USA).
Methods
Cell culture and preparation of cellular and mitochondrial extracts.
T-RexTM HeLa cells were stably transfected with the vector pcDNA™4/TO without insert (control) or with an insert coding for the NDPK-D WT or the NDPK-D mutants (Hl 5 IN and R90D) as described (7, 8). HeLa clones, grown in MEM medium as described (7), overexpress comparable levels of NDPK-D proteins already without specific induction. Maximal expression levels can be achieved by incubation with 1 pg/ml tetracycline for 24 h. For cell extract preparation, cells were grown in 3.5 cm diameter Petri dishes or in 6 well plates, were rinsed twice with ice-cold PBS and lysed in 50 pl RIPA/well containing anti-proteases (Calbiochem, cocktail set III or Complete®, Sigma), anti -phosphatases (Sigma, cocktail n°2) and 1 mM EDTA. The lysate was either used immediately or frozen in liquid nitrogen and stored at 20°C until use. For citrate synthase activity measurements, the fresh lysate was sonicated for additional 5 sec at 50% power and centrifuged at 10,000xg for 20 min at 4 °C and the supernatant kept. Crude HeLa mitochondria were isolated by differential centrifugation according to Eskes et al. (12). The protein concentration was determined by a BCA protein assay (Pierce), using bovine serum albumin (BSA) as standard.
MDA-MB-231, ZR75-1 and HBL100 cell lines were cultured in DMEM containing 10% fetal bovine serum (FBS). BT-474, BT-549, HCC-1428, MDA-MB-468 cells were grown in RPML1640 medium containing 10% FBS and 100 U/mL penicillin and 100 pg/mL streptomycin (P/S). HCC-1143, HCC-1187, HCC-1599, HCC-1500, and HCC-1937 cells were grown in RPMI-1640 medium containing 10% FBS, P/S, 1.5 g/L sodium bicarbonate, 10 mM Hepes and 1 mM sodium pyruvate. T47D cells were grown in RPMI-1640 medium containing 10% FBS, P/S and 0.2 U/mL bovine insulin. BT-483 cells were grown in RPMI-1640 medium containing 20% FBS, P/S and 0.01 mg/mL bovine insulin. MCF-10A, MCF-10-2A, and 184B5 cells were grown in DMEM-F12 containing 5% horse serum, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin and 500 ng/mL hydrocortisone. MCF-12A cells were grown in DMEM-F12 containing 5% horse serum, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 1.2 g/L sodium bicarbonate, 0.5 mM sodium pyruvate and 15 mM Hepes. HMEC and hTERT-HMEl cells were grown in Mammary Epithelial Cell Growth Medium BulletKit (Lonza, Basel, Switzerland). Hs578T and MDA-MB- 361 cells were grown in DMEM containing with 10% FBS and P/S. BT-20 and MCF-7 cells were grown in MEM containing 10% FBS, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino-acids and 1 mM sodium pyruvate. MDA-MB-157 and MDA-MB-453 cells were grown in Leibovitz's L-15 medium containing 10% FBS, P/S and 10 mM Hepes. MDA-MB-415 cells were grown in Leibovitz's L-15 medium containing 15% FBS, P/S, 10 mM Hepes, 0.01 mg/mL insulin and 0.01 mg/mL glutathione. CAMA1 cells were grown in Eagle’s MEM containing 10% FBS and P/S. MDA-MB-435S cells were grown in Leibovitz's L-15 medium containing 10% FBS, P/S, and 0.01 mg/mL insulin. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.
Immunoblot analysis.
Proteins from cell extracts were electrophoretically separated on 10% or 12.5% SDS polyacrylamide gels and transferred onto Immobilon P membranes (0.1 pm, Millipore) for 2 h at 22 V in 10 mM CAPS buffer, pH 11, 10% methanol for NDPK-D, as described in (7), or onto nitrocellulose membranes for 90 min at 50 V in 0.025 M Tris-base, 0.192 M glycine, 20% methanol, and 0.02% SDS for the other proteins. The polyclonal anti-NDPK-D was diluted 1/7500, the anti-a tubulin (loading control) 1/5000, and the other primary antibodies 1/500. Blots were revealed with appropriate peroxidase-coupled secondary antibodies and ECL Plus substrate (GE Healthcare).
Cell dispersion, aggregation, invasion and migration assays.
Cellular spatial distribution was characterized and quantified using algorithmic programs of cellular sociology based on the use of three previously described geometrical models, namely Voronoi's partition, Delaunay' graph and minimum spanning tree (MST) as described (13, 14). The aggregation assay was performed as reported (15) by seeding cells on top of a gelified agar medium. Aggregate formation was scored under an inverted microscope at xlO magnification after 24 h incubation at 37 °C. Native type I collagen invasion assays were performed as described earlier (14, 16, 17. Two- and three-dimensional migration assays are described in (13, 14) and (18), respectively.
Cell dispase assay.
Confluent monolayers of cells were washed with ice-cold PBS and separated from the plates by incubation with PBS free of Ca2+ and 0.6 U/mL of dispase I (MP Biomedicals, Irvine, CA, USA) for 35 min at 37°C. The dispase solution was removed by centrifugating the cells for 2 min at 400xg and replaced by 200 pL of PBS. The cells were mechanically separated by pipetting up and down five times with a 200 pL pipette. The aggregates were observed by light microscopy using the 10X objective (Echo Rebel Microscope, San Diego, CA, USA). The size of the aggregates was measured using the Fiji software (aggregates < 200 pm2 were excluded from the quantification). Wound healing assay.
Cells were grown until confluence on 24 well plates in DMEM supplemented with 10% FBS and antibiotics. Cells were starved for 24 h in DMEM without FBS and treated for 2 h with Cytosine P-D-Arabinofuranoside (AraC) to inhibit cell proliferation during the experiment. After starvation, cells were scratch-wounded using a sterile 200 pL pipette tip and suspended cells were removed by washing with PBS twice. The progress of cell migration into the wound was monitored every 24 h until wound closure using the 10X objective of an Echo Rebel Microscope. The bottom of the plate was marked for reference, and the same field of the monolayers were photographed immediately after performing the wound (time = 0 h) and at different time points after performing the scratch.
Matrix metalloprotease activity by gelatin zymography.
Culture media were collected and concentrated using 10 KDa cut-off ultra-centrifugal filter units (Amicon, Merck-Millipore, Burlington, MA, USA). Protein concentration was determined by the Bradford method, and 200 pg of concentrated supernatant proteins were assayed for proteolytic activity on gelatin-substrate gels. Briefly, samples were mixed with nonreducing loading buffer containing 2.5% SDS, 1% sucrose and separated in 8% acrylamide gels co-polymerized with 1 mg/mL gelatin. Electrophoresis was conducted at 80 V for 2.5 h, then the gels were rinsed twice in 2.5% Triton X-100, and then incubated in 50 mM Tris-HCl pH 7.4 and 5 mM CaCh assay buffer at 37 °C for 24 h. Gels were fixed and stained with 0.25% Coomassie Brilliant Blue G-250 in 10% acetic acid and 30% methanol. Proteolytic activity was detected as clear bands against the background stain of undigested substrate in the gel. Quantification was performed using ImageJ2 software (NIH, Bethesda, MD, USA).
Enzyme activities.
NDPK activity in mitochondrial extracts (1-10 pg protein/assay) was measured spectrophotometrically by a coupled pyruvate kinase-lactate dehydrogenase assay using 0.2 mM ATP and 0.2 mM TDP as substrates and adding 100 pM Ap5A to inhibit endogenous adenylate kinase, as described previously (6,7). As an estimate of mitochondrial mass, citrate synthase (CS) activity was measured in cell lysates in the presence of 150 mM Tris pH 8, 150 pM 5, 5'-dithiobis-(2 -nitrobenzoic acid) (DTNB), 300 pM acetyl-coenzyme A and 500 pM oxaloacetate. Reduction of DTNB by CS at 37°C was followed spectrophotometrically at 412 nm and CS activity calculated in nkat/mg of total protein.
Rael pulldown assay.
Cells were seeded overnight and starved in low serum for 24 h then washed twice with ice cold PBS supplemented with MgC12 (25 mM) and lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 25 mM MgC12, 500 mM NaCl, 2 mM sodium pyrophosphate, 1 mM NaVO4, 2 mM phenylmethyl sulphonyl fluoride (PMSF), 10 pg/mL aprotinin, 10 pg/mL leupeptin). Lysates were clarified by centrifugation, and equal amounts of protein lysates were incubated with 20 pL of purified glutathione S-transferase (GST)-CRIB (Cdc42/Rac interactive binding motif) immobilized on glutathione-Sepharose beads (17-0756-01, GE Healthcare,) for Ihour rocking at 4°C. The beads were washed 3 times in lysis buffer and boiled for 10 min in SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 0.002% bromophenol blue, 2% SDS, and 5% P-mercaptoethanol). The samples were analysed for Rael pull-down by Western Blot using an anti-Racl antibody.
Mitochondrial network analysis.
For cellular staining of mitochondria, cells cultivated on microscope glass slides were fixed in 3.7% paraformaldehyde, permeabilized in PBS containing 0.5% Triton, blocked in PBS containing 3% BSA, and incubated with anti -mouse MnSOD primary antibody (dilution 1 : 150) and with donkey anti-mouse IgG secondary antibody, Alexa Fluor 488-conjugate (dilution 1 : 150). Slides were examined with a Leica HC microscope. For mitochondrial staining with a mitochondrion- selective dye and NDPK-D immunodetection, cells were incubated with 50 nM MitoTrackerTM Red CMXRos (Molecular Probes) for 30 min at 37°C before fixation, treatment as described above and incubation with anti-NDPK-D affinity -purified antibody (dilution 1 :500) followed by the Alexa-Fluor 488-conjugated secondary anti-rabbit antibodies. Before examination, slides were mounted with Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA).
Fixed cells immunostained for mitochondrial MnSOD were subjected to image analysis with Image J software (NIH) to extract foreground and segment mitochondria using an adaptive threshold (“top-hat filtering”). In the resulting binary image of the mitochondrial network, regions of interest (ROI) were selected in peripheral regions of cells, where individual mitochondrial elements can be more easily detected as compared to the mitochondrial clusters close to the nucleus. Morphometric analysis of each ROI was done with Volocity v.4.0.1 software (Improvision, France), which yielded values for average length and the elongation factor, calculated as (mean_perimeter2)/(4 it mean area). To visualize the mitochondrial network in vivo, Hela cells grown on Labtek® plates (Nunc) were labeled with 100 nM MitoTracker Green FM (Invitrogen) for 20 min at 37°C. Images were acquired with a confocal laser-scanning microscope (Leica TCS SP2 AOBS; 488 nm excitation, 500-550 nm detection), equipped with a perfusion chamber (LaCon) and an incubation system (O2-CO2-°C, PeCon). Confocal pinhole (set to 1 Airy) and all other parameters were kept constant for all experiments, and images taken from randomly chosen fields containing 8-12 cells.
Mitochondrial and glycolytic function.
Mitochondrial membrane potential was determined with about 106 HeLa cells per sample, first incubated for 30 min at 37°C with 50 nM TMRM (tetramethylrhodamine, methyl ester Life Technologies, ThermoFisher Scientific, Waltham, Massachusetts, US), a membrane potential sensitive dye, and 100 nM Mitotracker GreenFM (Life Technologies, ThermoFisher Scientific, Waltham, Massachusetts, US). Cells were then centrifuged at 700 g for 4 min at 4°C, the pellet was resuspended in 1 ml PBS and TMRM fluorescence gated by Mitotracker signal was analyzed by FACS (BD LSR FORTESSA, Becton Dickinson, Le Pont-de-Claix, France). Cells were then incubated with 1 pl 50 mM CCCP for another 5 min at room temperature to entirely depolarize the mitochondria, and again analyzed by FACS. About 20000-50000 events gated on Mitotracker fluorescence were measured, and differences in samples before and after addition of CCCP calculated as readout for mitochondrial membrane potential. Laser excitation was 488nm and 532nm for Mitotracker GreenFM and TMRM, respectively. Fluorescence emission was collected with a 530/30 nm band-pass filter for Mitotracker GreenFM and 585/15 nm band-pass filter for TMRM.
Changes in mitochondrial membrane potential produced by NDPK-D knockdown in ZR75- 1 cells were determined with the tetramethylrhodamine ethyl ester (TMRE)-Mitochondrial Membrane Potential Assay Kit (Abeam) following the manufacturer’s protocol. Briefly, ZR- 75-1 cells were supplemented with 200 nM TMRE and incubated in the dark for 10 min at 37 °C. Then, the cells were trypsinized and washed three times with PBS. Fluorescence intensity of TMRE was measured by Spectrum Cellometer (Nexcelom Biosciences, Lawrence, MA) by setting the filter excitation at 502 nm and emission at 595 nm, as previously reported (36,37). Data was analyzed with FCS Express 7 (De Novo Software).
Oxygen consumption in intact HeLa cells was measured in a thermostatically controlled Clark electrode oxygraph at 37°C (Strathkelvin MS200A system). HeLa cells were detached by trypsin and counted. A cell suspension (100 millions of trypan blue negative cells per ml) was prepared in Roswell Park Memorial Institute (RPMI) medium (ThermoFisher, France). Five million cells were added in the oxygraph chamber containing RPMI medium to a final volume of 500 pl. Oxygen consumption of cells was measured with succinate as substrate before and after the addition of oligomycin (0.06 pg/ml) and FCCP (0.5pM), and results expressed as nmol O2 per minute per mg of cellular protein. Calcium retention capacity in digitonin permeabilized cells was determined in trypsin- detached HeLa cells (2 million trypan blue negative cells), permeabilized immediately before use by incubation under stirring for 2 min at 30°C in 250 mM sucrose, 10 mM Tris-MOPS, 1 mM Pi -Tris (pH 7.4) supplemented with 100 pg/ml digitonin. Initially, 0.25 pM Calcium Green-5N (Molecular Probes, Eugene, OR, USA) are added, followed by 5 mM succinate, to a final volume of 1 ml. The calcium retention capacity was measured by sequential addition of 12,5 pM Ca2+ pulses until permeability transition occurs (34). Extramitochondrial Ca2+ was measured fluorimetrically at 30°C using a PTI Quantamaster C61 spectrofluorimeter (excitation: 506 nm; emission: 530 nm) (35). Results are expressed as nmol Ca2+ per 2 millions of trypan blue negative cells.
Extracellular acidification rate (ECAR) to estimate glycolytic activity was determined by an Agilent Seahorse XF flux analyzer according to manufacturer’s instructions.
ROS and oxidative stress.
ROS production was detected using the dye CM-H2DCFDA. Cells were incubated with CM-H2DCFDA (9 pM) in DMEM without FBS. Quantification was performed with a plate fluorescence reader (Spectrafluor Plus, Tecan-France, Trappes, France) at 520 nm (19). Further markers of oxidative stress were analyzed as described in (20), including protein oxidation by thiols groups (SH) (21) and Ferric Reducing Ability of Plasma (FRAP) by ferric reduction (22). The lipid hydroperoxides were determined using a lipid hydroperoxide assay kit (Cayman Chemical Co., USA) according to the manufacturer’s instructions.
To quantify changes in oxidative stress produced in the mitochondria, cells were incubated with 5 pM MitoSOX™ reagent dissolved in DMSO, following the manufacturer’s instructions. After 10 min incubation at 37 °C in the dark, the cells were trypsinized and washed three times with PBS. Fluorescence was recorded using the Spectrum Cellometer (Nexcelom Biosciences, Lawrence, MA, USA) with excitation/emission maxima of 510/580 nm. Data was analyzed with FCS Express 7 (De Novo Software).
Measurement of intracellular nucleotides by LC-HRMS.
Cell extracts were prepared by cell lysis with the cold mixture methanol/water (70/30, v/v) after removing of cell medium and two washes with cold PBS. Extracts were stored at -80°C before analysis. Analysis of nucleoside mono-, di- and triphosphates in cell extracts was performed on an Ultimate 3000 liquid chromatography system (ThermoFisher Scientific™, Bremen, Germany) coupled with a Q-Exactive Plus Orbitrap mass spectrometer (ThermoFisher Scientific™, Bremen, Germany) using a validated method (38). Results were expressed as the ratio of triphosphate/diphosphate which corresponds to area of the nucleoside triphosphate peak/area of the nucleoside diphosphate peak and as the ratio of diphosphate/monophosphate which corresponds to area of the nucleoside diphosphate peak/area of the nucleoside monophosphate peak.
Proliferation Assays.
Cell proliferation was examined in real time using the xCELLigence RTCA MP System (Roche Applied Science). HeLa clones were seeded at 5000 cells/well into 96-well plates and proliferation was continuously monitored every hour over a time period between 12 and 36 hours. Data analysis was performed using RTCA 1.2 software supplied with the instrument. Levels of proliferation markers, cyclin A, cyclin Bl and PCNA were analyzed by Western blotting of HeLa clone extracts.
2D-DIGE proteomic analysis.
Sample preparation for 2D-electrophoresis. Cells were grown in 6 cm diameter Petri dishes close to confluence (106 cells), washed in cold PBS, harvested with a rubber policeman and pelleted by centrifugation at 800xg for 5 min. Pellets, dried by aspiration, were frozen in liquid nitrogen and stored at -80 °C until use. Pellets were lysed and homogenized 20 min on ice in 100 pL of UTCD buffer (8 M urea, 2 M thiourea, 4% CHAPS and 50 mM dithiothreitol (DTT)). The lysates were centrifuged at 20,000xg, at 4 °C for 1 h. The supernatants were collected and proteins were precipitated with a 2-D Clean-Up Kit (GE Healthcare) following the manufacturer's instructions. The pellets were solubilized in 100 pL of UTC buffer (UTCD buffer without DTT) and the protein concentration determined using Quick- Start Bradford Dye Reagent (Bio-Rad).
Two-dimensional differential in-gel electrophoresis (2D-DIGE). Three independent samples of two independent clones for each condition (control HeLa-Trex cells transfected with empty vector (CTR1A, B, C; CTR2B, C, D); cells overexpressing the wild-type NDPK-D (WT1 A, B, C; WT2A, C, D), the catalytically inactive (KD1 A, B, D; KD2A, B, C) and the CL- binding-deficient enzyme (BD1A, B, C; BD2A, B, D) were analyzed by 2D-DIGE. 50 pg of proteins of each sample were labeled with Cy3 or Cy5 CyDye™DIGE Fluor minimal dyes (GE Healthcare) following the manufacturer's instructions. The internal standard (IS) was prepared by mixing equal amounts of each sample and labeled with Cy2. 50 pg of labeled samples (Cy3 or Cy5) and internal standard (Cy2) were mixed in twelve different combinations as follows: WTlA-Cy3/CTRlA-Cy5/IS-Cy2, WT2A-Cy3/CTR2B-Cy5/IS-Cy2, KD2A-Cy3/WT2C- Cy5/IS-Cy2, WTlB-Cy3/KDlA-Cy5/IS-Cy2, BDlA-Cy3/WT2D-Cy5/IS-Cy2, BD2A- Cy3/WTlC-Cy5/IS-Cy2, KDlB-Cy3/CTRlB-Cy5/IS-Cy2, CTR2C-Cy3/KD1D-Cy5/ IS-Cy2, BDlB-Cy3/KD2B-Cy5/IS-Cy2, KD2C-Cy3/BD2B-Cy5/IS-Cy2, CTRlC-Cy3/BDlC-Cy5/IS- Cy2 and CTR2D-Cy3/BD2D-Cy5/IS-Cy2. Each of the twelve mixes (150 pg) was analyzed by 2D-DIGE as previously described with minor modifications (39). Protein separation was performed by isoelectrofocusing on 18-cm pH 3-11NL Immobiline™ Drystrips (IPG strips, GE Healthcare) in the first dimension and SDS-PAGE on twelve different 8 to 18% acrylamide gels in the second dimension. Cy2, Cy3, and Cy5 components of each gel were individually imaged as described previously (39).
Statistical analysis. Image analysis, relative quantification of spot intensity, statistical evaluation using one-way ANOVA followed by a Tukey’s multiple comparison test and PC A (principal component analysis) were carried out with DeCyder 7.2 software (GE Healthcare). Normalization across all gels was performed using the internal standard. A spot was considered as differentially represented between two sample groups if the following conditions were fulfilled: p-value below 0.05 and protein abundance fold change above +1.3 or below -1.3.
Protein identification by Mass Spectrometry (MS) and database searching. For MS identification of proteins of interest, two distinct semi-preparative 2D-gels were prepared using 400 pg of WT and 400 pg of a mix of BD and KD, respectively, to rehydrate the IPG strips. After electrophoresis, 2D-gels were fixed and stained as described in (40). Gels were scanned using a Typhoon 9400 Trio Variable Mode Imager (GE Healthcare) at 488/520 nm, 100 pm resolution. Spots of interest were excised using the Ettan spot picker (GE Healthcare). In-gel digestion was carried out with trypsin, according to a published procedure with minor adjustments (41) and using for all steps a Freedom EVO 100 digester/spotter robot (Tecan, Switzerland). For MS and MS/MS ORBITRAP, analyses were performed using an Ultimate 3000 Rapid Separation Liquid Chromatographic (RSLC) system (Thermo Fisher Scientific) online with a hybrid LTQ-Orbitrap-Velos mass spectrometer (Thermo Fisher Scientific). The Linear Trap Quadrupole Orbitrap mass spectrometer acquired data throughout the elution process and operated in a data dependent scheme with full MS scans acquired with the Orbitrap, followed by up to 20 LTQ MS/MS CID spectra on the most abundant ions detected in the MS scan. The fragmentation was permitted for precursors with a charge state of 2, 3, 4 and above. For the spectral processing, the software used to generate mgf (Mascot generic format) files was Proteome discoverer vl.4.0.288. The threshold of Signal to Noise for extraction values is 3. Database searches were carried out using Mascot version 2.4 (Matrix Science, London, UK) on “homo sapiens” proteins (20,345 sequences) from the SwissProt databank containing 542,503 sequences (192,888,369 residues) (February 2014). The search parameters were as follows: carbamidomethylation as a variable modification for cysteines, and oxidation as a variable modification for methionines. Up to 1 missed tryptic cleavage was tolerated, and mass accuracy tolerance levels of 10 ppm for precursors and 0.45 Da for fragments were used for all tryptic mass searches. Positive identification was based on a Mascot score above the significance level (i.e. 5%).
RNA interference.
Two specific siRNAs targeting NME1 (Sil 5’-GGCUGUAGGAAAUCUAGUU - SEQ ID NO:3; Si2 5’-GGAUUCCGCCUUGUUGGUC - SEQ ID NO:4) or targeting NME4 (Sil 5’ -AGCACAAGAUUGGACCAAU - SEQ ID NO:5; Si2 5’ GCAAGAACCCAAGCCCACA - SEQ ID NO:6) synthesized by ThermoFisher Scientific (ThermoFisher Scientific, Waltham, MA, USA) were used. The siRNA control sequence was 5’-GGCUGUAGAAGCUAUAGUU (SEQ ID NO:7). Cells were transfected with control or specific siRNA sequence using the DharmaFECT 4 transfection reagent (Dharmacon, Inc, Lafayette, CO, USA).
Experimental metastasis assays.
All the animal experiments were carried out at NCI (Frederick, MA, USA) under an approved NCI-Animal Use Agreement. HeLa clones stably expressing different constructs (CTR1, CTR2, WT1, WT2, KD1, KD2) were trypsinized, washed, and resuspended in PBS and injected into the lateral tail vein (n=9 for each group) of six-week-old Balb/c athymic nude female mice (1 x 106 HeLa cells per injection). Thirteen weeks post-injection, at necropsy, the lungs were collected and fixed in Bouins' solution. Lung metastatic lesions were counted using H&E section and reported as a mean for each group.
RT-qPCR (HeLa cell lines).
Quantitative PCR was performed on HeLa stable cell lines using the mix 2X Roche LightCycler (480 SY Green Master Mix- ref 4 887 352 001- Roche Diagnostics, Mannheim, Germany) on a Light Cycler 96 Real Time PCR Roche (Roche Diagnostics). Data from each sample were normalized on the basis of its content in HPRT (hypoxanthine-guanine phosphorribosyl transferase) transcripts. The primers used were: ISG15, S10A4, FSCN1, N-cadherin, SYUG, HPRT, CDH2. Data were collected and analyzed with Roche LightCycler® 96 System Software 3.5.3 (Roche Diagnostics). Data were expressed as a relative amount (2'AACT) of a control experiment used as a calibrator.
Breast cancer cohort.
Primary breast tumors were obtained from 526 women treated at Institut Curie - Hopital Rene Huguenin (Saint-Cloud, France) between 1978 and 2008.. All patients have given their approval for the potential use of their tumor samples for scientific purpose. This study was approved by the local ethics committee (Breast Group of Institut Curie - Rene Huguenin Hospital). The samples were immediately stored in liquid nitrogen until RNA extraction. A tumor sample was considered suitable for this study if the proportion of tumor cells exceeded 70%. All patients (mean age 60.9 years, range 29 - 91 years) met the following criteria: primary unilateral non-metastatic breast carcinoma for which complete clinicopathological data and follow-up were available; no radiotherapy or chemotherapy before surgery; and full follow-up at Institut Curie - Hopital Rene Huguenin. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2) statuses were determined at the protein level by biochemical methods (Dextran-coated charcoal method, enzyme immunoassay or immunohistochemistry) and confirmed by real-time quantitative RT-PCR. The population was divided into four groups according to HR (ER and PR) and ERBB2 statuses as follows: two luminal subtypes [HR+ (ERa+ or PR+)ZERBB2+ (n=58)] and [HR+ (ERa+ or PR+)ZERBB2- (n=294)]; an ERBB2+ subtype [HR- (ERa- and PR-)/ERBB2+ (n=73)] and a triple-negative subtype [HR- (ERa- and PR-)/ERBB2- (n=101)].
RT-qPCR (breast cancer samples).
Samples of the breast tumor cohort (526 human clinical specimens) have been analyzed in RT-qPCR. Conditions for total RNA extraction, cDNA synthesis and PCR reaction have been described elsewhere (23). Quantitative values were obtained from the cycle number (Ct value) using ABI Prism 7900HT Sequence Detection System and PE Biosystems analysis software according to the manufacturer’s instruction (Perkin-Elmer Applied Biosystems, Foster City, CA). Data from each sample were normalized on the basis of its content in TBP transcripts. TBP encoding the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) was selected as an endogenous control due to the moderate level of its transcripts and the absence of known TBP retro-pseudogenes (retro-pseudogenes lead to coamplification of contaminating genomic DNA and thus interfere with RT-PCR transcripts, despite the use of primers in separate exons). Results, expressed as N-fold differences in target gene expression relative to the TBP gene and termed ‘Ntarget’, were determined as Ntarget = 2ACtsample, where the ACt value of the sample was determined by subtracting the average Ct value of target gene from the average Ct value of TBP gene.
METABRIC and TCGA databases.
Gene expression data were extracted from cBioPortal for Cancer Genomics (https://www.cbioportal.org/), which provides visualization, analysis, and download of large- scale cancer genomics data sets (42,43), by specifically focusing on METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) (44,45) and TCGA (The Cancer Genome Atlas) research network database. EMT signature was calculated with the methodology defined in (46). Survival distributions was obtained from http : //kmpl ot . com/ analy si s .
Statistical analysis.
Statistical analyses were performed using GraphPadPrism (version 7.00) software. The comparisons of NME4 mRNA levels between the different subgroups of human breast tumors and the comparisons of lung metastases number between the different CTR,WT and KD clones in immunocompromised mice, were performed by the Kruskal -Wallis test followed by two by two comparison performed with the Dunn’s test. Relationships between mRNA expression of the different target genes from the human breast tumor cohort (n=526 human breast tumor clinical specimens) and from the TCGA databank were identified using the non-parametric Spearman’s rank correlation test (relationship between two quantitative parameters). Linear regression analysis with ANOVA test was performed to determine significance for correlations between different genes from the METABRIC databank. Survival distributions were estimated with the Kaplan-Meier method and the significance of differences between survival rates was ascertained with the log-rank test. For all other comparisons between two groups, we performed an unpaired Student’s t-test. Differences were considered significant at confidence levels greater than 95% (p<0.05).
Results:
NDPK-D mutations induce a morphotypic switch linked to a loss of intercellular adhesion
The HeLa clones that are analyzed here in detail have been used already in our earlier studies (7, 8, 10). The control HeLa clones contained empty vector (control, abbreviated as CTR) and expressed low levels of endogenous NDPK-D (data not shown). Clones stably transfected with vectors for different NDPK-D variants, namely wild-type (WT), CL-binding deficient (R90D mutation or BD) or kinase-dead (Hl 5 IN mutation or KD), expressed high levels of these NDPK-D proteins, presenting as a single strong band at the size of mature enzyme (data not shown). In addition, in case of WT and BD clones, also high NDP kinase enzyme activity was detectable in mitochondria while the activity of the catalytically inactive mutant was barely detectable (data not shown). Since the protein precursor is inactive (6), this further indicates correct mitochondrial import and processing of the pre-proteins. Exclusive localization of all NDPK-D proteins within mitochondria, and their absence in the cytosol, was validated by immunocytochemistry (data not shown). This confirms our earlier data with overexpression of GFP -fused protein in HEK293, subcellular fractionation (6,7) and immunocytochemical localization of these NDPK-D variants in HeLa (7,8,10). All this demonstrates correct processing and mitochondrial import of NDPK-D variants. Of note NDPK-A (NME1) and NDPK-B (NME2) protein levels remained unchanged in HeLa clones (data not shown).
The most obvious difference immediately observable between the HeLa clones were two distinct and very different types of cell cohesion and morphology (data not shown). While controls and NDPK-D WT expressing cells were organized as epithelioid clusters, even more compact for the WT clone, cells expressing either of the two NDPK-D mutants, R90D or Hl 5 IN, grew as randomly dispersed single cells, exhibiting none to very few cell-cell contacts, most pronounced for the Hl 5 IN mutant (data not shown). Cell cohesion was further quantified in a cell dispersion assay, using algorithmic programs of cell sociology (13). This analysis confirmed that expression of mutated NDPK-D resulted in a more random spatial distribution of the cells compared with controls and WT enzyme expressing cells (data not shown).
The acquisition of a scattered cellular phenotype is often associated with alterations in intercellular adhesion. We therefore investigated the impact of NDPK-D mutations on the cellcell adhesion properties of HeLa cells in a slow aggregation assay (15), where cells are grown on soft agar (data not shown). Again, control and NDPK-D WT expressing clones formed compact aggregates, whereas both mutant clones did not.
Cell-cell adhesion of HeLa cells relies entirely on N-cadherin, since the well-known epithelial marker E-cadherin is not expressed (24). N-cadherin was markedly decreased in both mutant NDPK-D expressing clones as compared to control and WT NDPK-D expressing cells, again most pronounced for the kinase-dead Hl 5 IN mutant (data not shown). All these data consistently show a clear and similar morphotypic switch that occurred in the mutant NDPK-D expressing clones, with a marked loss of cell-cell aggregation and cell-cell adhesion.
NDPK-D mutations increase 2D and 3D cell migration
To further examine consequences of altered cell morphology and cell-cell adhesion induced by NDPK-D mutants, we applied different migration assays (data not shown). In the 2D assay, trajectories of WT NDPK-D expressing clones were restricted and random, while a striking directional migration was observed with control and mutant expressing clones (data not shown). Overexpression of WT NDPK-D significantly reduced the 2D migration speed as compared to controls and mutants (data not shown). The 2D migration speed was significantly increased in kinase-dead mutant expressing clones (KD) as compared to wild type NDPK-D (WT) expressing clones (data not shown). The increased 2D migration speed exhibited by the mutant HeLa clones is also obvious through examination of video microscopy images (data not shown). Similarly, the 3D migration assay revealed higher migration speed along the x-y-z planes for the two mutant expressing clones (KD1, KD2) as compared to the WT NDPK-D expressing clone (data not shown). Since cell migration is largely mediated by Rho-GTPases, we evaluated Rael activation in these Hela clones via GST-pulldown assays (data not shown). As compared to controls, overexpression of WT NDPK-D tended to reduce active, GTP -bound Rael, while overexpression of kinase-dead NDPK-D strongly increased Racl-GTP levels. Similar changes were observed for phosphorylation of p21-activated kinase PAK1, a downstream Rael effector. These data suggest that the Rael pathway participated in increased migration of this mutant.
NDPK-D mutations increase the 3D invasive potential
Because the capacity to breach extracellular matrix barriers is critical for metastasis, we assessed whether expression of NDPK-D mutants affects the ability of HeLa cells to invade a three-dimensional matrix of native type I collagen during 24 hours (data not shown). HeLa cells are notoriously poor in degrading the extracellular matrix (25). When seeded on native type I collagen, mutant NDPK-D formed numerous cellular protrusions, which invaded the collagen layer, while controls and WT enzyme expressing cells presented only few of these (data not shown). Expression of both NDPK-D mutants strongly increased invasion through native type I collagen as compared to WT NDPK-D; the latter was even significantly lower as compared to the control (data not shown). This is reminiscent to siRNA knock-down of cytosolic NDPK- A/NME1/NM23-H1, a confirmed metastasis suppressor, which also generates a scattered (data not shown) and highly invasive phenotype (data not shown), reaching an invasion index of 20% through native type I collagen, similar to NDPK-D loss-of-function mutants. This indicates similar anti-invasive functions of NDPK-D/NME4/NM23-H4 and NDPK-A/NME1/NM23-H1 in HeLa cells. In addition, NME1 silencing induced activation of the Rael signaling network, similar to NDPK-D loss-of-function mutants (data not shown). The invasive phenotype of mutant NDPK-D expression was further confirmed by a 14-day invasion assay. Here, sections of the collagen layer were examined two-weeks after seeding the HeLa clones. While the WT clones remained on the surface, the kinase-dead clones deeply penetrated into the collagen layer (data not shown). The invasive program of mutant clones was not related to an advantage of proliferation since their proliferation rates were lower than the one of the wild-type clones, also confirmed by protein levels of proliferation markers such as cyclin A, cyclin Bl, and PCNA that were higher in WT clones than in CTR, BD and KD clones (data not shown).
Selective pharmacological inhibition of pro-invasive pathways, including PI3K, Src, p38, JNK, and epidermal growth factor receptor (EGFR), strongly reduced invasion of a type I collagen matrix by both mutant NDPK-D (data not shown). Stimulation of EGFR and its downstream signaling (ERK, Akt, GSK3B) by EGF was largely reduced in WT NDPK-D cells as compared to controls, while activation in NDPK-D mutants was comparable to controls or even higher (data not shown). Thus, strong responsiveness of mutant clones to EGF correlates with their reduced invasive potential upon EGFR inhibition.
The cellular proteome reveals changes in metastasis-related and mitochondrial proteins The morphotypic switch and the scattered/migratory/invasive phenotype observed for Hela cells expressing NDPK-D mutants are striking features, considering that they are triggered by a single point mutation in a mitochondrial protein. This implies communication between molecular NDPK-D structure/function and cellular behavior in respect to cell adhesion, motility and invasive potential. Since this should be mediated by changes in the cellular proteome, we next performed a comparative 2D-DIGE proteomic study with two independent clones of every experimental group (CTR, WT, KD and BD NDPK-D). To identify significant changes within the differential expression pattern, multiple group-to-group comparisons were performed using the DeCyder biological variation analysis module (data not shown). A total of 206 differentially expressed protein spots were identified by mass spectrometry, corresponding to 157 different proteins (data not shown). Importantly, most changes in protein abundance relative to WT NDPK-D expressing cells occurred in the same sense for both mutants, the kinase-dead KD (data not shown) and the cardiolipin-binding deficient BD (data not shown). For quantitative analysis, principal component analysis was performed on the entire set of detected protein spots. Clearly, all samples of clones expressing mutant NDPK-D segregated from samples of WT NDPK-D expressing cells, while the control samples were different from both of the former groups (data not shown). Although the two clones analyzed for each of the four experimental groups also segregated to a certain degree, they remained quite close, emphasizing the good reproducibility of phenotypes and 2D-DIGE analysis. The proteomic profiles recapitulate the similarities seen for the scattered and migrative/invasive phenotypes. They suggest that both NDPK-D mutations trigger similar pathways to acquire these phenotypes, acting via a change in the cellular protein expression and/or degradation program.
Analysis of functionally related groups of proteins with IPA software (QIAGEN Redwood City, www.qiagen.com/ingenuity) identified more than twenty proteins involved in metastasis (data not shown). The proteins are named according to their Entry Names in UniProt (data not shown). They showed a change in expression level between wild type (WT) vs control (CTR) and mutant (BD and KD) NDPK-D expressing clones. Of the overexpressed proteins involved in metastasis, fifteen were found in both mutant clones, the kinase-dead KD and the cardiolipin-binding deficient BD; only one was unique to KD (AK1C3, abbreviated according to UniProt) and one to BD (TCTP). Among these proteins, the strongest overexpression was found for y-synuclein (SYUG: +7.8, +10.8; all data given as fold-changes in KD and BD vs. WT). Further overexpressed proteins included actin-bundling protein Fascin (FSCN1 : +2.3, +2.3), two calcium-binding S100 proteins (S10A4: +3 to +5; SIOAB: about +2), cytoskeletal tubulin P-2A (TBB2A: +3.9, +3.8) and interferon-stimulated gene 15 (ISG15: +8.1, +4.4). The latter, although not assigned to the metastatic pathway by IP A, was recently reported to promote invasion (26). Of the downregulated proteins, again six were found in both mutant clones, and only one was unique to BD (ROAA). The down-regulation was less marked. Of note, downregulation of N-cadherin (data not shown) failed to be identified by the proteomic analysis, probably due to its low pl (4.6) and high Mr (100 kDa). Immunoblotting analysis confirmed the 2D-DIGE results, e.g. overexpression of Fascin, y-synuclein, ISG15, S100A4 (S10A4) and tubulin P-2A in KD vs. WT (data not shown). At the mRNA level (data not shown), consistent with these changes in protein abundance, we observed strong up-regulation of ISG15, S100A4, and SYUG. As expected, N-cadherin mRNA was downregulated in the kinase-dead KD clones as compared to WT cells. This suggests that these proteins are mainly regulated at the transcriptional level. FSCN1 mRNA levels were unchanged, indicating a different regulation. In conclusion, coordinated deregulation of multiple metastasis-related proteins in both NDPK- D mutant-expressing clones provides a molecular rationale for a role of NDPK-D in the metastatic process.
Another functional group identified by IPA was Mitochondrial Dysfunction and Oxidative Stress (data not shown). Indeed, among proteins differentially expressed in mutant KD and BD clones vs. WT were many mitochondrial proteins. A marked change was downregulation of several core subunits of ATP synthase: alpha (ATPA: -1.5, -1.7), beta (ATPB: -2.0, -1.9) and delta (ATP5H: -1.4, -1.6), while few changes were detected in the respiratory chain. These concerned complex I, with a downregulation of the core subunit NADH-ubiquinone oxidoreductase 75 kDa (NDUS1, -1.7, -1.6) in the matrix-facing dehydrogenase module of the peripheral arm, and upregulation of the accessory subunit NADH dehydrogenase 1 alpha subcomplex subunit 8 (NDUA8, +1.7, +1.7), which faces the intermembrane space and is essential for complex I assembly (27, 28). The most downregulated mitochondrial protein (>2-fold) was carbamoylphosphate synthase 1 (CPSM), catalyzing the first committed step leading to arginine biosynthesis and urea cycle. This protein is represented by several spots resulting probably of maturation and/or posttranslational modifications (29). Interestingly, there was also down-regulation of two other mitochondrial proteins that could potentially compensate for NDPK-D functions: adenylate kinase 3, a GTP: AMP phosphotransferase (KAD3: -1.6, -1.4) able to generate GTP from GDP and ADP, and MICOS complex subunit MIC60 (IMMT : -1.8, -1.7). The latter complex bridges inner and outer mitochondrial membrane, similar to NDPK-D, and organizes cristae (30-32). Finally, within the family of voltage-dependent anion channels (VDACs), controlling among others outer mitochondrial membrane permeability, an isoform switch occurred, with upregulated isoform 3 (VDAC3: +2.4, +1.3) and downregulated isoform 1 (VDAC1 : -1.5, -1.5) and isoform 2 (VDAC2: -1.4, -1.6). Based on these data, and the phenotype of NDPK-D mutant expression described herein, we hypothesized (i) that there should be a primary effect of NDPK-D mutations on mitochondrial structure and/or function, and (ii) that this effect should be again similar for both mutants.
NDPK-D mutations affect mitochondrial structure and function
Given its mitochondrial localization, we suspected that NDPK-D loss-of-function has primary effects on mitochondria. We first studied the mitochondrial network of HeLa cells, fixed and immunostained for the mitochondrial protein Mn-superoxide dismutase (MnSOD; data not shown). Both NDPK-D mutant clones showed fragmentation of the network as compared to WT and control cells, determined by decreased filament length, elongation, surface area, and interconnectivity (data not shown). In contrast, the WT clone had higher elongation and surface area parameters as compared to controls. Thus, high levels of wild-type NDPK-D led to the most connected, filamentous mitochondrial network, while expression of NDPK-D mutants led to mitochondrial fragmentation, consistent with the key role of NDPK-D in fueling the mitochondrial fusion protein OPA1 (9). Similar networks were observed with MitoTracker Green live stained cells (not shown). Correlated with fragmentation, NDPK-D mutant clones also had lower mitochondrial mass as compared to WT and control cells, consistent with a preferential elimination of fragmented, smaller mitochondria (data not shown).
We then determined basic functional parameters of mitochondria. The average mitochondrial membrane potential (A m) decreased mainly in the NDPK-D mutant clones, giving first evidence for some mitochondrial dysfunction (data not shown). Next, activity of the Krebs cycle enzyme citrate synthase (CS) increased with overexpression of WT NDPK-D, but decreased with the loss-of-function mutants as compared to controls (data not shown). These changes cannot be explained by altered mitochondrial mass, thus indicating some rewiring of Krebs cycle activity in mutant vs. WT NDPK-D clones, consistent with a decreased abundance of the key Krebs cycle enzyme isocitrate dehydrogenase in mutant clones (IDH3A: -1.6, -1.5) in 2D-DIGE. Respiratory parameters of intact cells were analyzed by oxygraphy. Basal respiration and total electron transfer capacity after uncoupling with CCCP (data not shown, (33)) were reduced in both mutant NDPK-D clones as compared to the WT NDPK-D clone and controls, reflecting reduced mitochondrial content (data not shown). Leak respiration, i.e. basal uncoupling of mutant clones was less reduced, significant only relative to controls. Per mitochondrial mass, leak respiration even increased in the kinase dead KD mutant (not shown), consistent with its decreased membrane potential. The capacity of mitochondria to accumulate calcium without opening the mitochondrial permeability transition pore (mtPTP) is another global readout of mitochondrial function (data not shown). This calcium retention capacity, determined in di gitonin permeabilized HeLa cells, was unchanged at baseline (except for the BD mutant) and with mtPTP inhibition by cyclosporine A (data not shown). However, mtPTP inhibition by rotenone, an inhibitor of respiratory complex I (34, 35), alone (not shown) or in combination with cyclosporine A (data not shown), was reduced in both mutant NDPK-D clones as compared to the WT and controls. The specificity of the effect for rotenone suggests altered mtPTP regulation at the level of complex I (35).
The glycolytic activity of NDPK-D mutant clones at baseline and after inhibition of mitochondrial ATP synthesis as determined from the extracellular acidification rate (data not shown) were both increased as compared to the control and WT NDPK-D clones (data not shown). This is consistent with a compensatory up-regulation of glycolytic ATP generation due to reduced respiratory ATP synthesis in the NDPK-D mutant clones. We therefore investigated consequences of this metabolic switch on cell energetics. Based on a full quantification of adenine, guanine, cytosine and uracil nucleotides (data not shown), overall nucleotide equilibria like ATP/ADP and GTP/GDP ratios as well as ATP/AMP and GTP/GMP ratios were not significantly altered in NDPK-D mutants (data not shown). However, induction of mild energy stress was apparent by activation or overexpression of kinases that are involved in cellular energy homeostasis (data not shown). The energy sensor AMP-activated protein kinase (AMPK) was phosphorylated and activated in BD and KD clones relative to WT, also observed with phosphorylation of the AMPK substrate acetyl-CoA carboxylase in BD clones (data not shown). The mitochondrial isoform of creatine kinase (umtCK) was upregulated in both BD and KD clones relative to WT, and the mitochondrial adenylate kinase AK2 was upregulated in the BD clone only (data not shown). Upregulation of these kinases in the mitochondrial intermembrane space often occurs as a compensatory response under energy stress (31).
Finally, we were interested whether the observed functional changes would affect the cellular levels of reactive oxygen species (ROS) (data not shown). Indeed, increased ROS generation was observed in both mutants as compared with the WT expressing clone. The latter has a decreased ROS level as compared with control (data not shown). This in agreement with the measurement of markers of peroxidation. Oxidation of proteins (reduced thiols) was increased in mutants relative to control and wild-type (data not shown). Lipid peroxides were barely detectable in wild-type expressing cells as compared with control. In both mutants a significant increase in lipid peroxides was observed (data not shown). The kinase-dead (KD) clone also had reduced antioxidant capacity (data not shown).
NDPK-D is a gatekeeper against EMT in breast cancer cells
To investigate the general relevance of NDPK-D for EMT, invasion, and metastasis, we turned to human breast cancer. We first analyzed NME4 transcript levels by RTqPCR of a panel of human breast tumor cell lines according to their normal-like, hormone receptor (HR)- positive, and triple-negative (HR- and HER2-negative) status, where the HR-positive subtype has a more favorable prognosis than the triple-negative subtype (data not shown). We observed significantly more NME4 mRNA in the HR-positive human breast tumor cell lines than in the normal-like cell lines; these levels significantly decreased in the triple-negative human breast tumor cell lines, reaching a similar level to that observed in normal-like cell lines (data not shown).
For genetic manipulation of functional NDPK-D levels in breast cancer, we chose two of these human breast tumor cell lines, MDA-MB-231 and ZR75-1. The former had the lowest level of NME4 mRNA and was highly invasive and metastatic, while the latter had the highest level of NME4 mRNA and was minimally invasive with an epithelial-like phenotype (data not shown). We overexpressed WT and both BD and KD mutants of NDPK-D in MDA-MB-231 cells, similar to our approach with HeLa cells. The control MDA-MB-231 clones containing empty vector (CTR) expressed undetectable levels of endogenous NDPK-D (data not shown). Clones stably transfected with vectors for WT, BD or KD NDPK-D expressed high levels of these proteins, presenting as a single strong band at the size of mature enzyme (data not shown). As shown for HeLa clones (data not shown), MDA-MB-231 clones exhibited an immunostaining with anti-NDPK-D antibodies strictly colocalizing with mitochondria- selective marker (data not shown). With ZR75-1 cells, we did the contrary experiment, depleting NDPK-D specifically by expressing two different siRNAs. Western blotting confirmed the effective siRNA-mediated knockdown of NDPK-D (data not shown). We then investigated in both cell lines the functional consequences of such genetically modified NDPK- D expression on cell-cell adhesion, migration and invasion properties. First, we applied a wound-healing assay, where a confluent cell monolayer is breached and the degree of migration to close the wound in a given time period is determined (data not shown). In case of MDA-MB-231 cells, two different clones for each condition were studied. When comparing the wounds immediately after the scratch (0 h) and 24 h later, control, BD and KD cells completely closed the wound, while WT cells were unable to do so, leaving 20- 40% of the original scratch wound surface uncovered (data not shown). In the contrary experiment with ZR75-1 cells, a model of slow growth breast carcinoma, wound closure was analyzed for 120 h after scratching (Figure 1A). Here, cells depleted of NDPK-D migrated faster and covered nearly 100% of the scratch wound area at 96 h. Migration of control cells expressing NDPK-D was significantly slower than that of the knockdown cells and they were unable to close the wound at 96 h.
We also assessed whether expression of NDPK-D variants affects the ability of MDA-MB- 231 cells to invade a three-dimensional matrix of native type I collagen during 24 hours (Figure IB). Expression of WT NDPK-D strongly reduced invasion through native type I collagen as compared to CTR; the latter had an invasive potential similar to that of both NDPK-D mutants. We then analyzed a global readout of cell migration and invasion, the secretion and activation of the metalloproteinases MMP2 and MMP9 (Figure 1C). Cell migration requires cyclic formation and destruction of focal adhesions and alteration of the composition of the extracellular matrix (47). Migrating cells achieve this process by secreting Zn2+-dependent MMPs that respond to growth factors, cytokines and hormones (48,49). The MDA-MB-231 clones overexpressing WT NDPK-D as compared to control clones showed a decrease by 60% and 80% in the gelatinase activity of MMP9 and MMP2, respectively (Figure 1C), consistent with their impaired wound healing. No significant changes in MMP activity was detected in cells expressing BD or KD NDPK-D. In the contrary experiment with NDPK-D depletion in ZR75-1 cells, we found that NDPK-D depletion increased the secretion of MMP9 by 1.5-fold (data not shown), consistent with accelerated wound healing in this case. MMP2 activation was undetectable in these cells.
We then studied cellular aggregation by disrupting cell-cell contacts with the protease dispase (Figure 2A, 2B and data not shown). Consistent with a highly invasive phenotype, control MDA-MB-231 clones as well as BD and KD clones rendered a low number of aggregates with a diameter over 200 pm2 (Figure 2A). In contrast, clones overexpressing WT NDPK-D presented enhanced cell-cell adhesion properties with over 100 aggregates that largely exceeded 200 pm2, with sizes even beyond 1000 pm2, and a mean area of 600 pm2 in both WT clones (Figure 2A). Conversely, siRNA-mediated knockdown of NDPK-D in ZR75- 1 cells resulted in decreased adhesive capabilities compared to controls expressing NDPK-D (Figure 2B). Aggregates of cells transfected with the scramble siRNA presented average areas of 600 pm2, whereas those of NDPK-D-depleted cells had only few aggregates over 200 pm2 (Figure 2B).
Finally, we tested the effect of NDPK-D depletion on mitochondrial function and oxidative stress. Mitochondrial membrane potential, A m, was reduced about 20% in NDPK-D-depleted ZR75-1 cells when compared to the control cells expressing NDPK-D (data not shown). Since mitochondrial dysfunction can result in oxidative stress, we used the mitochondrial superoxide fluorogenic indicator MitoSOX™ Red to selectively detect superoxide species in live cells. MitoSOX™ Red is oxidized by superoxide, resulting in red fluorescence. The ZR75-1 cells depleted of NDPK-D presented a small but significant increase in mitochondrial ROS when compared to control cells expressing NDPK-D (data not shown), potentially as a result of impaired mitochondrial function.
Taken together, results obtained with breast cancer MDA-MB-231 and ZR75-1 cells are consistent with our data on cervical cancer HeLa cells, namely showing increased cell motility, reduced cell-cell adhesion, and mitochondrial dysfunction with NDPK-D downregulation or loss-of-function mutations. This strongly supports our conclusion that NDPK-D expression is negatively associated with breast cancer progression and invasion.
Overexpression of NDPK-D reduces in vivo metastasis dissemination
As NDPK-D loss-of-function increases the invasive potential of HeLa cells in vitro, we used nude mice to test the role of NDPK-D for metastasis formation in vivo. We injected HeLa cells expressing the different NDPK-D species, empty vector (CTR1, CTR2), wild-type (WT1, WT2), and the kinase-dead mutant (KD1, KD2) via the tail vein of nude mice. After 13 weeks, we found that overexpression of the wild-type NDPK-D reduced pulmonary colonization as compared to empty vector expression condition (Figure 3). By contrast, overexpression of the kinase-dead NDPK-D mutant significantly augmented the number of lung metastases as compared to the wild-type NDPK-D overexpression. These data identify NME4 as a new metastasis suppressor gene.
NME4 expression is negatively associated with EMT and tumor invasion markers and is associated with beneficial clinical outcome in human cancer
Based on our novel findings on anti-invasive and anti-metastatic functions of NDPK-D, we predicted that its expression might be down-regulated in human aggressive tumors showing EMT and invasion in comparison to tumors with a good prognosis. We first determined mRNA levels of NME4, CDH1 and KRT18, the genes encoding NDPK-D and two well-known epithelial markers E-cadherin and cytokeratin 18, respectively, in an important cohort of 526 human breast tumors from patients with well-documented follow-up by using RT-qPCR (Table 1). Consistently, we found a strong positive association of NME4 with CDH1 and KRT18 (data not shown). Strikingly, NME4 mRNA levels in this cohort were the lowest in the most aggressive human breast tumors with worst prognosis, the so-called triple-negative breast tumors (data not shown). Similar associations were observed with another metastasis suppressor in this cohort : NME1 (data not shown).
We further analyzed mutations in the NME4 gene that are available in the TCGA human database, covering a large panel of different tumors. These data revealed that NME4 is very rarely mutated (data not shown). Importantly, we observed the same clinical phenotype with the well-known metastasis suppressor NME1. Indeed, in the cohort of 526 human breast tumors, a positive association was observed between NME1 and CDH1 and KRT18 and a negative one with the mesenchymal marker VIM, the vimentin gene (data not shown). Very interestingly, NME4 and NME1 transcript levels were themselves positively associated (data not shown). In addition, NME1, like NME4, is rarely mutated (data not shown). Using the same cohort, a positive association was observed between NME1 and CDH1 and KRT18 and a negative one with the mesenchymal marker VIM, the vimentin gene (data not shown). In addition, NME1, like NME4, is rarely mutated (data not shown).
To confirm these findings, we first interrogated the METABRIC database (a cohort of 1904 human breast tumors) for the relation between mRNA levels of NME4 and mRNA of epithelial and mesenchymal markers. Consistently, we found a positive association of NME4 with CDH1 and KRT18 (data not shown). We observed a negative association between NME4 and mesenchymal markers CDH2 and VIM (data not shown) as well as many EMT drivers like ZEB1, ZEB2, SNAI2, TWIST1 and TWIST2 (data not shown). The NME4 mRNA levels also negatively associated with the overall EMT score (data not shown). Next, in the TCGA cohort of 1084 human breast invasive carcinoma samples, we observed similar significant associations between mRNA levels of NME4 and markers of EMT and tumor invasion (data not shown); for example, concerning the EMT markers, a strong positive association between NME4 and the epithelial markers KRT18, KRT8 (the CK8 gene), JUP (the plakoglobin gene), TJP3 (the ZO-3 gene) and CLDN3 (the claudin 3 gene); a significant negative association between NME4 and the mesenchymal markers VIM, CDH2 and the EMT drivers like SNAI1, SNAI2, ZEB1, ZEB2. Concerning the tumor invasion markers, we observed an overall negative association between NME4 and the genes encoding proteases involved in matrix degradation including MMP7, ADAMI 7 and the genes encoding proteins involved in actin cytoskeleton remodeling such as ROCK1, ROCK2, LIMK2, CFL2, MYO5A. Similar associations have been observed with other claudins, MMPs including MMP14 (also called MT1-MMP which is essential for matrix degradation during tumor invasion), and ADAM proteins (data not shown).
We further interrogated the TCGA database on human cervical squamous cell carcinoma. This revealed similar associations, in particular positive association with epithelial markers (KRT18, KRT8, CLDN3) and negative association with EMT drivers (SNAI2 and ZER2). We also observed an overall negative association between NME4 and tumor invasion markers in this tumor type, in particular between NME4 n MMP14, the key player of invasion, and between NME4 and markers of invadopodia, the plasma membrane protrusions responsible of matrix degradation and enriched in MMP14 (data not shown). These data identify \owNME4 expression as associated with EMT and tumor invasion features as a generic trait in human clinical tumor samples.
To assess the prognostic value of NME4 expression in several human tumor cohorts, we interrogated the publicly available human cancer KM Plotter database that contain gene expression data and overall survival information stratifying patient samples into groups of low and high expression. The outcome was then compared between low and high NME4 expression groups in eight different tumor types. In six carcinoma types (breast, ovarian, lung, pancreatic, uterine, and esophageal carcinoma), low expression of NME4 was associated with a poor prognosis (Figure 4A,B,C,D,E,F); this is also the case for tumors other than carcinomas such as pheochromocytoma, paraganglioma and sarcoma (Figure 4G,H). Taken together, these data show that positive association of NME4 expression with beneficial clinical outcome is a generic trait in cancer.
Conclusion
Collectively, these data reveal a prominent role of altered NDPK-D in crucial features of cancer metastasis such as loss of intercellular adhesion, migration, invasion, and EMT. In fine, they suggest a communication between mitochondria, cytosol and nuclear genes for a pro- metastatic reprogramming of cellular protein expression as the driving force towards the observed morphotypic switch. Definitely, these in vitro, in vivo, and clinical findings show for the first time that NME4 is a new metastasis suppressor gene, another member of the NME/NDPK superfamily proteins and the first one localized in mitochondria, and that NME4 has the potential of being a strong prognostic biomarker. In perspective, targeting dysregulated mitochondrial fission/fusion dynamics may provide a novel strategy for inhibiting cancer metastasis REFERENCES:
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Claims

CLAIMS:
1. A method for predicting the survival time of a patient suffering from a cancer, comprising i) determining the expression level of NDPK-D in a sample obtained from the patient ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is higher than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is lower than its predetermined reference value.
2. A method for predicting the invasiveness of a cancer, comprising i) determining in a sample obtained from the patient the expression level of NDPK-D ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is higher than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is lower than its predetermined reference value.
3. The method according to claims 1 or 2 wherein the sample can be blood, peripheral - blood, serum, plasma, tumoral circulating cells or a tumor sample.
4. The method according to claim 3 wherein the sample is a tumor sample.
5. The method according to claim 3 or 4 wherein the sample is a solid tumor sample.
6. A fusion protein comprising the protein according to the invention that is fused to at least one heterologous polypeptide.
7. A NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression for use in the treatment of cancer in a subject in need thereof.
8. The NDPK-D protein or fragment thereof or a fusion protein thereof and/or the agent for NDPK-D protein expression for use according to claim 7 wherein the cancer is a metastatic cancer.
9. The NDPK-D protein for use according to claims 7 or 8 wherein the NDPK-D protein comprises a sequence as set forth by SEQ ID NO: 1.
10. A method for treating a cancer in a subj ect in need thereof comprising the administration to said subject the NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression.
11. The method of claim 10, wherein the agent for NDPK-D protein expression is a vector encoding NME4, NME4 mRNA or is the NME4 mRNA molecule itself.
12. The method according to claims 1 or 2 or the NDPK-D protein or fragment thereof or the fusion protein thereof and/or the agent for NDPK-D protein expression for use according to claim 7 wherein the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.
13. The method according to claim 12 or the NDPK-D protein or fragment thereof or the fusion protein thereof and/or the agent for NDPK-D protein expression for use according to claim 12 wherein the cancer may be a breast, ovarian, lung, pancreatic, uterine or esophageal carcinoma or a pheochromocytoma, a paraganglioma or a sarcoma.
14. A pharmaceutical composition comprising A NDPK-D protein or fragment thereof or a fusion protein thereof and/or an agent for NDPK-D protein expression for use in the treatment of a cancer in subject in need thereof.
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