WO2018141753A1 - Procédé de traitement de carcinomes à cellules squameuses - Google Patents

Procédé de traitement de carcinomes à cellules squameuses Download PDF

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WO2018141753A1
WO2018141753A1 PCT/EP2018/052308 EP2018052308W WO2018141753A1 WO 2018141753 A1 WO2018141753 A1 WO 2018141753A1 EP 2018052308 W EP2018052308 W EP 2018052308W WO 2018141753 A1 WO2018141753 A1 WO 2018141753A1
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scc
cells
elmol
tgfbr2
metastasis
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Géraldine GUASCH
Heather MCCAULEY
Véronique Chevrier
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique (Cnrs)
Institut Jean Paoli & Irene Calmettes
Université D'aix Marseille
University Of Cincinnati
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Publication of WO2018141753A1 publication Critical patent/WO2018141753A1/fr

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    • C07K14/475—Growth factors; Growth regulators
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
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    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present invention relates to an agonist of the TGFP signaling pathway for use in the treatment of squamous cell carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC metastasis derived from a squamous cell carcinomas
  • Transition zones are distinct anatomical regions between two different types of epithelia, and function as stem cell niches in many regions of the body [Runck LA, et al, 2010].
  • stem cell characteristics such as label-retention and self-renewal, and expression of stem cell markers such as the cell surface glycoprotein CD34, have been found in the transition zones between the cornea and conjunctiva in the eye [Zieske JD 2010] between the esophagus and the stomach [Kalabis J et al, 2008] between the endocervix and ectocervix [Elson DA et al, 2000], between the mesothelium and oviductal epithelium of the ovary [Flesken-Nikitin A et al, 2009] and between the anal canal and the rectum [Runck LA, et al, 2010].
  • Transition zones are uniquely susceptible to carcinogenesis, and the resulting tumors are typically highly malignant and associated with poor prognosis [Roman AKS et al, 2011 ] .
  • Ocular surface squamous neoplasias are relatively rare, but most of them involve the limbus.
  • the transition zone in the ovary was recently shown to be acutely sensitive to oncogenic transformation.
  • Cervical cancers arise at the transition between the columnar epithelium of the endocervix and the squamous epithelium of the ectocervix [Elson DA et al, 2000].
  • Highly malignant squamous cell carcinomas develop between the stratified epithelium of the anal canal and the simple epithelia of the rectum [Grodsky L., 1961].
  • TGFP signaling seems to be a hallmark of aggressive transition zone cancers.
  • mutations or loss of TGFP downstream effectors SMAD2 and SMAD4 are common [Maliekal TT et al, 2003], and loss of nuclear SMAD2 and SMAD4 is associated with poor survival [Kloth JN et al, 2008].
  • TGFP receptor (TGFpRII) protein expression is decreased and loss of phosphorylated SMAD2 is observed, even at early stages, suggesting that loss of TGFP signaling may be an early event in carcinogenesis [Guasch G et al, 2007].
  • Mouse models targeting components of the TGFP signaling pathway have been generated [Munoz NM et al, 2006]. Many epithelia develop normally despite the loss of a component of the TGFP signaling pathway [Guasch G et al, 2007 and Munoz NM et al, 2006].
  • tumorigenesis occurs rapidly when these epithelia are exposed to carcinogens, polyomavirus middle T antigen expression, oncogenic mutations, such as mutations in APC, or activated H-ras or K-ras, or spontaneously in transition zones.
  • loss of SMAD3 [Nam KT et al, 2012] or BMP signaling [Bleuming SA et , 2007] results in invasive carcinoma.
  • Mice with a neuronal-specific deletion of Tgfbrl develop spontaneous periorbital and perianal SCC [Honjo Y et al, 2007].
  • mice lacking Tgfbr2 in all Keratin 14-expressing (K14+) progenitors of the stratified epithelia is morphologically normal, but these mice develop spontaneous SCC in cervical and anorectal transition zones [Guasch G et al, 2007].
  • RHO and RAC-guanine triphosphatases are small G proteins (21-25kDa), and belong to the RAS superfamily. They act as molecular switches to elicit rapid changes in cell shape, polarity, and migratory ability in response to external cues [Parri M et al, 2010] and are major players in malignant cell invasion.
  • RAC exists in an inactive form, bound to GDP, and in an active form, bound to GTP [Parri M et al, 2010].
  • Guanine exchange factors GEFs
  • GAPs GTPase activating proteins
  • GEFs act downstream of many signaling pathways, including growth factor receptors, integrins, cadherins, and cytokine receptors [Parri M et al, 2010].
  • Engulfment and cell motility (ELMO) proteins (originally described as CED-12 in C. elegans) participate in RAC 1 -dependent engulfment and apoptosis [Cote J-F et al, 2007 and Gumienny TL et al, 2001].
  • ELMO proteins form a complex with DOCK proteins that serves as a GEF for RAC proteins. This complex plays important roles in chemotaxis, phagocytosis, neurite outgrowth, and cancer cell invasion [Laurin M et al, 2014 and Cote J-F et al, 2007].
  • CSCs cancer stem cells
  • Tgfbr2 deficient anorectal SCC In this study, the inventors used the murine Tgfbr2 deficient anorectal SCC model to study the consequences of loss of TGFP signaling in CSC-driven tumor propagation and metastasis. They found that these Tgfbr2 cKO anorectal SCC, which spontaneously metastasize to the lungs, contain a unique population of epithelial cells with features of CSCs, including: expression of the CSC marker CD34, clonogenicity in vitro, tumorigenicity in vivo, and upregulation of genes associated with invasion and metastasis.
  • the present invention relates to the present invention relates to an agonist of the TGFP signaling pathway for use in the treatment of squamous cell carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • the present invention relates to an agonist of the TGFP signaling pathway for use in the treatment of squamous cell carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • TGFP signaling pathway has its general meaning in the art and denotes a signaling pathway involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions. In spite of the wide range of cellular processes that the TGFP signaling pathway regulates, the process is relatively simple. TGFP superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4.
  • R-SMADs receptor-regulated SMADs
  • R-SMAD/coSMAD complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression. More particularly, the inventors described a novel mechanism by which loss of TGFP receptor II (Tgfbr2) mediates invasion and metastasis through de-repression of ELMO 1. Particularly, they showed that restoration of Tgfbr2 results in a complete block of ELMO 1.
  • the agonist of the TGFP signaling pathway may be an agonist of Tgfbr2 or an antagonist of ELMO 1 or an inhibitor of the expression of ELMO 1.
  • the invention also relates to an agonist of Tgfbr2 for use in the treatment of squamous cell carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC metastasis derived from a squamous cell carcinomas
  • the invention also relates to an antagonist of ELMO 1 or to an inhibitor of the expression of ELMO 1 for use in the treatment of squamous cells carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cells carcinomas
  • the invention also relates to an antagonist of ELMO 1 or to an inhibitor of the expression of ELMO 1 for use in the treatment of squamous cells carcinomas (SCC).
  • SCC squamous cells carcinomas
  • the invention relates to an antagonist of ELMO 1 or to an inhibitor of the expression of ELMOl for use in the inhibition of squamous cells carcinomas invasion.
  • the invention relates to an antagonist of ELMOl or to an inhibitor of the expression of ELMOl for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • the invention relates to an antagonist of ELMOl or to an inhibitor of the expression of ELMOl for use in the treatment of an aggressive or highly aggressive squamous cells carcinomas (SCC) and/or for use in the inhibition of aggressive or highly aggressive squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from an aggressive or highly aggressive squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC aggressive or highly aggressive squamous cells carcinomas
  • SCC aggressive or highly aggressive squamous cells carcinomas
  • SCC squamous cell carcinomas
  • Tgfbr2 for "transforming growth factor, beta receptor ⁇ " has its general meaning in the art and denotes a member of the serine/threonine protein kinase family and the TGFB receptor subfamily.
  • the encoded protein is a transmembrane protein that has a protein kinase domain, forms a heterodimeric complex with another receptor protein, and binds TGF-beta. This receptor/ligand complex phosphorylates proteins, which then enter the nucleus and regulate the transcription of a subset of genes related to cell proliferation.
  • ELMOl for "Engulfment and cell motility protein 1” has its general meaning in the art and denotes a protein which interacts with the dedicator of cyto- kinesis 1 protein to promote phagocytosis and effect cell shape changes. Alternative splicing of this gene results in multiple transcript variants encoding different isoforms.
  • An exemplary sequence for human ELMOl gene is deposited in the Entrez database under accession numbers 9844.
  • An exemplary sequence for human ELMOl protein is deposited in the UniProt database under accession numbers Q92556.
  • squamous cell carcinomas or “SCC” also known as squamous cell cancer has is general meaning in the art and denotes a cancer that begins from squamous cells, a type of skin cell. It is one of the main types of skin cancer. SCC constitute by far the largest number of cancers that develop in humans. Cancers that involve the eyes, anus, cervix, head and neck, and vagina are also most often squamous cell cancers. The esophagus, urinary bladder, prostate, and lung are other possible sites.
  • the SCC can be a SCC of transition zones, can be a SCC of transition zones involving cancer stem cells (CSCs) and can be selected in the group consisting in anorectal, eyes, cervix, head and neck, esophagus and stomach cancers.
  • CSCs cancer stem cells
  • the invention relates to an antagonist of ELMOl or to an inhibitor of the expression of ELMOl for use in the treatment of anorectal, eyes, cervix, head and neck, esophagus or stomach cancers and/or for use in the inhibition of anorectal, cervix, head and neck, eye, esophagus or stomach cancers cells invasion and/or for use in the treatment of metastasis derived from eye, anorectal, cervix, head and neck, or esophagus, stomach cancers in a subject in need thereof.
  • 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 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 a 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 compound according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • the antagonist according to the invention is an antibody.
  • Antibodies or directed against ELMOl can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against ELMOl can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Ko filer and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti- ELMOl single chain antibodies.
  • Coumpounds useful in practicing the present invention also include anti-ELMOl antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to ELMOl .
  • Humanized anti- ELMOl antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibody according to the invention may be the ab 174298 sell by Abeam.
  • the antibody according to the invention is a single domain antibody against ELMOl and particularly against ELMOl .
  • the term "single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDRs complementarity determining region
  • CDR complementarity determining region
  • VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • VHH variants and modified form thereof may be produced under any known technique in the art such as in- vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • immobilized antigen e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • VHHs from immune libraries are attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals.
  • the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the "Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • the "Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • a single domain antibody against ELMOl may be used to treat a squamous cell carcinomas (SCC), to inhibit a squamous cells carcinomas invasion and to treat metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC metastasis derived from a squamous cell carcinomas
  • the compound according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the compound according to the invention is a polypeptide.
  • the polypeptide is an antagonist of ELMO 1 and is capable to prevent the function of ELMO 1.
  • the polypeptide can be a mutated ELMOl protein or a similar protein without the function of ELMOl .
  • polypeptide of the invention may be linked to a cell-penetrating peptide" to allow the penetration of the polypeptide in the cell.
  • 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).
  • polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell.
  • a host cell Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • 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.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability 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.
  • PEG Polyethylene glycol
  • Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • PEGylation techniques for the effective modification of drugs.
  • drug delivery polymers that consist of alternating polymers of PEG and tri- functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.).
  • the PEG chains typically 2000 daltons or less
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half- life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold- limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • a specific trigger typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the ELMOl inhibitor according to the invention is an inhibitor of ELMOl gene expression.
  • Small inhibitory RNAs can also function as inhibitors of ELMOl expression for use in the present invention.
  • ELMOl gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ELMOl gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • Ribozymes can also function as inhibitors of ELMOl gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of ELMOl mRNA sequences are thereby useful within the scope of the present invention.
  • Specific ribozyme cleavage sites within any potential R A target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of ELMO 1 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half- life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing ELMOl .
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen- encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes
  • a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • an siRNa or a shRNA or a ribozyme against ELMOl may be used to treat a squamous cell carcinomas (SCC) and/or to inhibit a squamous cells carcinomas invasion and/or to treat metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC metastasis derived from a squamous cell carcinomas
  • the shRNA use to inhibit the expression of ELMOl may have the sequence CCATCTTATACGACTCAAATT (SEQ ID NO: 1).
  • an shRNA having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% of homology with the shRNA of SEQ ID NO: 1 can be used to to treat a squamous cell carcinomas (SCC) and/or to inhibit a squamous cells carcinomas invasion and/or to treat metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC squamous cell carcinomas
  • a test is necessary. For that purpose, we can quantify by flow- cytometry the presence of metastases in the lung and by histology we can predict the invasiveness of the cancer stem cells.
  • An antagonist or inhibitor of ELMOl will block cancer cell migration in vitro and can be measured by wound healing assay. In this kind of assay, 5xl0 4 cells are plated in ibidi culture insert onto the 24-well plate (ibidi cat-80241) for 24h to reach 90-95%) of confluence. A wound is created by removing inserts.
  • mice bearing tumors that can be followed with a fluorescent reporter will be treated with the inhibitor and lungs will be dissociated into a single cell suspension
  • Whole lungs will be sorted for YFP and analyzed using a FACS Aria II (BD Biosciences) and FACSDiva software (BD Biosciences).
  • Percent YFP+ cells will be calculated by dividing number of YFP+ cells by total cells after gating for appropriately sized single cells by forward scatter and side scatter.
  • Another object of the invention relates to a method for treating a squamous cell carcinomas (SCC) and/or for inhibiting a squamous cells carcinomas invasion and/or for treating metastasis derived from a squamous cell carcinomas (SCC) comprising administering to a subject in need thereof a therapeutically effective amount of an agonist of the TGFP signaling pathway.
  • SCC squamous cell carcinomas
  • SCC squamous cell carcinomas
  • the invention in another embodiment, relates to a method for treating squamous cell carcinomas (SCC) and/or for inhibiting squamous cells carcinomas invasion and/or for treating metastasis derived from a squamous cell carcinomas (SCC) comprising administering to a subject in need thereof a therapeutically effective amount of to an agonist of Tgfbr2.
  • SCC squamous cell carcinomas
  • SCC squamous cell carcinomas
  • the invention in another embodiment, relates to a method for treating squamous cell carcinomas (SCC) and/or for inhibiting squamous cells carcinomas invasion and/or for treating metastasis derived from a squamous cell carcinomas (SCC) comprising administering to a subject in need thereof a therapeutically effective amount of an antagonist of ELMO 1 or of an inhibitor of the expression of ELMO 1.
  • another compound used to usually treat squamous cell carcinomas can be used in combination with an agonist of the TGFP signaling pathway and/or particularly with an antagonist of ELMOl and/or an inhibitor of the expression of ELMOl .
  • SCC squamous cell carcinomas
  • Fluorouracil (5-FU), imiquimod or radiotherapy may be used in combination with an agonist of the TGFP signaling pathway.
  • Another object of the invention relates to a therapeutic composition comprising an agonist of the TGFP signaling pathway according to the invention for use in the treatment of squamous cell carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC metastasis derived from a squamous cell carcinomas
  • the invention relates to a therapeutic composition
  • a therapeutic composition comprising an antagonist of ELMOl or of an inhibitor of the expression of ELMOl according to the invention for use in the treatment of squamous cell carcinomas (SCC) and/or for use in the inhibition of squamous cells carcinomas invasion and/or for use in the treatment of metastasis derived from a squamous cell carcinomas (SCC) in a subject in need thereof.
  • SCC squamous cell carcinomas
  • SCC squamous cell carcinomas
  • 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 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.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.
  • anti-cancer agents may be added to the pharmaceutical composition as described below.
  • Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide
  • Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levami
  • 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 agents 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.
  • the further therapeutic active agent can be 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 non- opioid 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.
  • buspirone and benzodiazepines
  • benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Knockdown of Elmo 1 in vitro affects cell migration.
  • Tgfbr2 flox/flox x K14-Cre mouse model has been derived in a pure C57BL/6N background and backcrossed into a mouse reporter containing an Enhanced Yellow Fluorescent Protein gene (eYFP) inserted into the Gt(ROSA)26Sor locus and called R26R-eYFPfiox-STOP-fiox (Jackson Laboratory).
  • Control mice were either Tgfbr2 flox/flox x R26R-eYFP flox - STOp - flox or Tgf r2 +/+ x R26R-eYFP flox - STOp - flox K14-Cre, all in a C57BL/6N background.
  • Transplantation assays were carried out in homozygous Nu/Nu female mice, approximately six to eight weeks old, as previously described.
  • the chemical mutagen 7,12- dimethyl-benz[a] anthracene (DMBA) was administered topically to the backskin of cKO mice for 16 weeks as previously described [Guasch G et al, 2007].
  • Mice are housed in a sterile barrier facility as previously described. All experiments were approved by the Cincinnati Children's Hospital Research Foundation Institutional Animal Care and Use Committee and carried out using standard procedures. The identification of each allele was performed by PCR on DNA extracted from clipping the ear of the mice as previously described [Guasch G et al, 2007].
  • Tumors were dissociated into a single cell suspension according to protocol established in our lab.
  • Cells in suspension were labeled with the following antibodies at the dilutions indicated: PE-Cy7 conjugated to rat-anti-mouse CD l ib (BD Pharmingen, 1/200), PE-Cy7 conjugated to rat-anti-mouse CD31 (BD Pharmingen 1/100), PE-Cy7 conjugated to anti-mouse CD45 (eBioscience 1/200), PE conjugated to CD49f, (BD Biosciences, 1/50), Pacific Blue conjugated to CD29 (Biolegend, 1/100), biotin conjugated to anti-mouse CD34 (eBioscience, 1/50) and APC conjugated to streptavidin (BD Pharmingen, 1/200).
  • RNA extraction 7-amino-actinomycin D (7-AAD, eBioscience, 20 ⁇ 1 0.05mg/ml stock per 106 cells) to exclude dead cells.
  • Tumor cell populations were sorted for R A extraction, tissue culture or transplantation using sterile practices using a FACS Aria II (BD Biosciences) and FACSDiva software (BD Biosciences) in the Research Flow Cytometry Core at CCHMC.
  • Cells isolated for RNA extraction were collected directly into cell lysis buffer containing beta- mercaptoethanol, vortexed and stored at -80°C until RNA extraction.
  • E media epithelial cell culture media(68)
  • E media epithelial cell culture media(68)
  • lOOORPM 0.05mM calcium
  • 4C resuspended in E media containing 0.3mM calcium
  • plated on a feeder layer of irradiated fibroblasts Cells isolated for transplantation were collected in E media without serum, centrifuged at lOOORPM for 5 minutes at 4C, resuspended in 30% Matrigel and transplanted as previously described [McCauley HA et al., 2013].
  • Tumors were dissected and portions of each tumor were processed for embedding in paraffin as well as embedding in OCT. Pieces of tumor were fixed in formalin for 24 hours at 4C, then dehydrated and embedded in paraffin in the Pathology Core Facility at CCHMC. Deparaffined sections were then rehydrated and stained with antibodies or Hematoxylin and Eosin in the Pathology Core at CCHMC.
  • pieces of tumor were fixed in 4% paraformaldehyde for 24 hours at 4C, then washed thoroughly in lx PBS and soaked in 30%> sucrose at 4C for 24 hours, then incubated in a slurry of 2: 1 fresh 30%> sucrose:OCT at 4C for 24 hours, then embedded in OCT compound (Tissue-Tek, Sakura, Torrance, CA) and stored at -80°C as previously described [McCauley HA et al., 2013].
  • Deparaffined tissue sections (5 ⁇ ) were subjected to antigen retrieval and immunostaining as previously described. Frozen tissue sections ( ⁇ ) were subjected to immunofluorescence labeling as previously described. Primary antibodies against the following proteins were used at the dilution indicated: green fluorescent protein, conjugated to Alexa Fluor 488 (Invitrogen, 1/1,000); a6 integrin/CD49f (BD Biosciences, 1/100), pi-integrin/CD29 (Millipore, 1/100), biotin conjugated to rat-anti-mouse CD34 (eBioscience, 1/50), Keratin-5 (Seven Hills Bioreagents, Rabbit 1/250 or Guinea Pig 1/5,000), ELMOl (Abeam, 1/100 for immunofluorescence, Sigma, 1/50 for immunohistochemistry), RAC1 (Cell Signalling, 1/100 and Santa-Cruz, 1/50), RAC2 (Millipore, 1/100), pSMAD2 (Cell Signaling Technology,
  • DAPI 4',6-diamidino-2-phenylindole
  • Tumor cells were sorted by FACS and CD34- and CD34+ epithelial populations were collected in E media containing 0.05mM calcium. Cells were centrifuged at lOOORPM for 5 minutes at 4°C, resuspended, and plated at equal densities on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) in E media containing 0.3mM calcium. MEFs were isolated from wild-type CD-I mice at embryonic day 13.5 and cultured in DMEM containing 10% serum and 1% penicillin- streptomycin.
  • MEFs irradiated mouse embryonic fibroblasts
  • CD34+ SCC cells were grown on plastic without feeders in E media containing 0.05mM calcium.
  • Wild-type anal keratmocytes were isolated from newborn C57BL/6 mice at postnatal day 1 by dissecting the anal canal, dissociating epidermis from dermis by incubating in dispase overnight at 4°C, extracting keratmocytes using 0.12% Trypsin- EDTA diluted in versene containing 0.1% glucose, and plating on a feeder layer of irradiated MEFs in E media containing 0.3mM calcium as described above. Keratinocytes were passaged to a new feeder layer of irradiated MEFs in E media containing 0.3mM calcium once confluent. After the third passage, wild-type anal keratinocytes were grown on plastic in E media containing 0.05mM calcium.
  • Western Blot Proteins were detected by Western blotting as previously described. Briefly, cells were lysed and proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblotting using antibodies to the following proteins at the dilutions indicated: p-Smad2 (Cell Signaling Technology, 1/1,000), c-Myc (Cell Signaling Technology, 1/1,000), Smad2/3 (BD Biosciences, 1/500), Keratin 8 (NICHD Developmental Studies Hybridoma Bank maintained by the University of Iowa, 1/1000), ⁇ -actin (Sigma, 1/2,000), RAC1 (Cell Signaling, 1/2000), ELMOl (AbCam, 1/2000), a-Tubulin (Sigma, 1/5000), GAPDH (Santa Cruz, 1/5000).
  • p-Smad2 Cell Signaling Technology, 1/1,000
  • c-Myc Cell Signaling Technology, 1/1,000
  • Smad2/3 BD Biosciences, 1/500
  • HRP-coupled secondary antibodies were used at 1/2,000 in 5% nonfat milk, and IRDye-conjugated secondary antibodies (Li-COR, Lincoln, NE) were used at 1/10,000 in 5% nonfat milk.
  • Immunoblots were developed using standard ECL (Amersham) and Luminata TM crescendo and classico (Millipore) as previously described(70) or the Odyssey CLx Infrared Imaging System (Li-COR, Lincoln, NE).
  • Reverse transcription (RT) reactions were diluted to lOng/ ⁇ and ⁇ of each RT was used for real-time PCR.
  • Real-time PCR was performed using the CFX96 real-time PCR System, CFX Manager Software and the SsoFast EvaGreen Supermix reagents (Biorad, Hercules, CA) or StepOne Plus real-time PCR system and the Power Sybr Green PCR Master Mix reagents (Applied Biosystems, Grand Island, NY). All reactions were run in triplicate and analyzed using the AACT method with relative expression normalized to Gapdh. Primers were designed for this experiment.
  • NIH3T3 cells were seeded in 10 cm plates at 80% confluence and transiently transfected with a pCMV-driven mouse SMAD3 (Sino Biological Inc., Daxing, China) using X-treme Gene transfection reagent (Roche Applied Science, Indianapolis, IN, USA) for 24 hours, then treated with recombinant human TGFpi (R&D Systems, Minneapolis, MN, USA, 2 ng/ml) for an additional 24 hours.
  • Cells were cross-linked with 1% formaldehyde and subjected to ChIP using an antibody against SMAD3 (Abeam, Cambridge, MA, USA) using a ChIP assay kit (Millipore, Billerica, MA, USA) according to manufacturer's instructions. After purification, DNA obtained from the ChIP assay was used as PCR templates to verify the interaction between DNA and protein, using primers designed to amplify distinct sites in the mouse Elmol and Dock2 promoters (using specific primers). PCR products were then subjected to gel electrophoresis on a 3% agarose gel using a molecular weight marker to verify the size of migrating bands.
  • the full length Mus musculus Tgfbr2 gene (1.7kb) was isolated from a pcDNA3 expression vector, sequenced and subcloned into the multi-cloning site of the pLVX-IRES-mCherry lentiviral vector (Clontech, Mountain View, CA) using the NEB Quick Ligation Kit (New England Biolabs, Ipswitch, MA), according to manufacturer's instructions. The resulting ligation was transformed into DH5a competent bacteria and selected on LB-amp plates overnight. DNA was extracted using a Maxi Prep DNA kit (Qiagen, Venlo, Limburg) according to manufacturer's instructions. Colonies were subjected to enzymatic digestion followed by sequencing to confirm the integration.
  • Vector control pLVX-IRES-mCherry
  • rescue pLVX-TGFpRII-IRES-mCherry
  • hairpin resistant ELMOl construct pLVX-IRES-mCherry-ELMOl * constructs were produced by the Cincinnati Children's Lentiviral Core and the lentivectors production facility/SFR Biosciences Gerland - Lyon Sud. 2mL of viral supernatant were used for each 10cm plate, after being washed in E Media with 0.05mM Ca++ and concentrated by three centrifugations at 4000rpm for 15 minutes at 4°C using a Vivaspin 20MWCO 30kDa column (GE Healthcare, Pittsburgh, PA).
  • Concentrated virus was combined with 8 ⁇ g/ml polybrene (Hexadimethrine bromide, Sigma. St. Louis, MO) and 3ml E Media with 0.05mM Ca++, applied to Tgfbr2 cKO CD34+ SCC cells seeded at 60%> confiuency in 10cm plates, and incubated at 37°C 5% C02 for 24 hours. 24 hours after infection, plates were washed three times with sterile IX PBS and given fresh E Media with 0.05mM Ca++. 48 hours after infection, YFP+mCherry+ cells were selected by FACS and used directly for in vivo orthotopic transplantation or re-plated for in vitro experiments.
  • TRCN0000112655, TRCNOOOOl 12656 Two MISSION shRNA pLKO. l-puro bacterial constructs against the mouse Elmol gene (TRCN0000112655, TRCNOOOOl 12656) and the SH02 control shRNA were purchased from Sigma Aldrich via the Cincinnati Children's Robotic Lenti-Library Core and lentivirus for SH02 control, TRCNOOOOl 12655 (ELMOl shRNA construct #1) and TRCNOOOOl 12656 (ELMOl shRNA construct #2) was produced by the Cincinnati Children's Lentiviral Core and the lentivectors production facility/SFR Biosciences Gerland - Lyon Sud.
  • Lungs from tumor-bearing mice were dissociated into a single cell suspension according to a modified protocol based on one previously established in our lab [McCauley HA et al, 2013]. Briefly, lungs were inflated with a cocktail containing dispase (Sigma, St. Louis, MO), 20% collagenase (Sigma, St. Louis, MO) and Hank's buffered saline solution (Gibco, Waltham, MA) and dissociated in the same cocktail for 30 minutes while shaking at 37°C. Dissociated lung tissue was washed and filtered according to our tumor dissociation protocol (47), with the added treatment of red blood cell lysis buffer according to manufacturer's instructions (eBioscience, San Diego, CA).
  • the wound-healing assay was used to determine cell migration ability. 5x104 cells were plated in ibidi culture insert onto the 24-well plate (ibidi cat-80241) for 24h to reach 90-95% of confluence. A wound was created by removing inserts. Cells were washed with PBS twice to remove detached cells and incubated with medium E low Ca2+ containing puromycin (1 ⁇ g/ml). The cells were observed under an inverted light microscope (Carl Zeiss) equipped with a CCD camera (Ropper) at XI 0 objective. Images were taken by MetaMorph software every 10 min for lOh. The wound widths of different area at each time points were measured with MetaMorph software. Data result from calculating the slope of linear trend curve of wound widths as a function of time and are representative of three experiments. Quantitative data are presented as the mean and significant difference was determined by two-tailed Student's t-test.
  • Cell culture dishes at 80 % confluence were washed with ice-cold PBS IX, lysed with 500 ⁇ of lysis buffer (50mM Tris, pH 7,2, 350mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, lOmM MgC12, protease inhibitor cocktail complete tablets (Roche)) and centrifuged for 5 min at 13,000RPM at 4C.
  • lysis buffer 50mM Tris, pH 7,2, 350mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, lOmM MgC12, protease inhibitor cocktail complete tablets (Roche)
  • the supernatant was incubated with bacterially produced glutathione- S -transferase (GST)-PAK-CD fusion protein, containing the Rac and Cdc42-binding region from human PAK1B (71) bound to glutathione-coupled Sepharose beads at 4C for 45 min.
  • GST glutathione- S -transferase
  • the beads and proteins bound to the fusion protein were washed three times with a wash buffer (50mM Tris, pH7.2, 1% Triton X-100, 150mM NaCl, 20mM MgC12, protease inhibitor cocktail complete tablets (Roche)) and eluted in Laemmli sample buffer (60mM Tris pH 6,8, 2% SDS, 10%> glycerine, 0,1 % bromophenol blue) and then analysed for bound Racl molecules by western blot using a Rac 1 antibody.
  • a wash buffer 50mM Tris, pH7.2, 1% Triton X-100, 150mM NaCl, 20mM MgC12, protease inhibitor cocktail complete tablets (Roche)
  • Laemmli sample buffer 60mM Tris pH 6,8, 2% SDS, 10%> glycerine, 0,1 % bromophenol blue
  • Cells were stained with 10 ⁇ Cell Proliferation Dye eFluor 670 (Affymetrix eBioscience) according to the manufacturer guideline and analyzed by flow cytometry at Oh, 24h, 48h and 72h with a Fortessa instrument (3 lasers 405/488/630) (Becton Dickinson). DAPI staining was used and excluded to acquire 104 cells in the live population. Three separate experiments have been done and the mean of the geometric mean for each sample has been calculated using Prism software.
  • Tgfbr2-deficient anorectal SCC contain a distinct population of cells with clonogenic potential
  • mice lacking Tgfbr2 in stratified epithelia expressing Keratin 14 develop spontaneous squamous cell carcinoma (SCC) at the transition zone between the anal canal and rectum [Guasch G et al, 2007].
  • SCC spontaneous squamous cell carcinoma
  • CSCs cancer stem cells
  • both YFP+CD34- and YFP+CD34+ cells sorted from Tgfbr2 cKO SCC formed colonies; however, CD34- colonies appeared to be differentiated paraclones and were unable to be passed more than once, whereas CD34+ colonies appeared to form holoclones, were able to proliferate extensively, and survived unlimited passage (data not shown).
  • CD34+ SCC cells did not respond to TGFP stimulation, confirming the loss of TGFpRII, and aberrantly expressed Keratin 8, a hallmark of SCC, compared to keratinocytes isolated from the anal canal of wild-type mice (data not shown).
  • Tgfbr2 cKO CD34+ SCC cells are enriched for in vivo tumorigenicity
  • the frequency of epithelial CD34+ cells in the secondary tumors ranged from 7% to 22%. CD34 expression within this population of cells was confirmed at the mRNA level (data not shown).
  • Tertiary anorectal tumors maintained the tumor hierarchy of the primary and secondary Tgfbr2 cKO SCC, and selection for CD34+ CSCs resulted in an increased ratio of CD34+ cells (75%) (data not shown).
  • CD34 was re- expressed in 49% of YFP+ epithelial tumor cells by FACS (data not shown), indicating that CD34 expression is dynamic in vivo. This is in accordance with the dynamic CD34 expression found in a DMBA-induced model of Tgfbr2 deficient SCC of the backskin.
  • CD34 remains a useful marker to assay the CSC properties of a population of cells isolated from murine SCC.
  • CD34+ cells which lack TGFP signaling, to form secondary and tertiary tumors that recapitulate the hierarchy of the Tgfbr2 cKO primary tumors suggests that CD34+ SCC cells are able to self- renew and differentiate in vivo.
  • Transcriptional profiling of anorectal CSCs identifies a metastatic signature and upregulation of RAC signaling at the invading front of Tgfbr2 cKO SCC
  • Tgfbr2 cKO SCC Squamous cell carcinomas, including those occurring at transition zones, frequently metastasize to the lung.
  • Tgfbr2 cKO SCC indeed spontaneously metastasized to the lungs with 100% frequency (n>30 mice analyzed) (data not shown).
  • These lung metastases expressed Keratin 5 (data not shown), indicating their squamous epithelial origin.
  • Tgfbr2 cKO lung metastases expressed YFP and contained populations of CD34+ cells (data not shown), recapitulating the hierarchy of the primary tumor of origin.
  • Upregulation of RAC signaling is unique to the anorectal Tgfbr2 cKO SCC and not found in Hras-induced skin SCC
  • EMT epithelial to mesenchymal transition
  • the GEF ELMOl is a novel target of TGFp signaling via SMAD3
  • ELMO proteins form a complex with DOCK proteins that serves as a GEF for RAC proteins in many cellular processes, including cancer cell invasion. Because Dock2 and Elmo 1 mRNA were both upregulated in Tgfbr2 cKO CD34 + SCC cells, we investigated whether the loss of TGFP signaling was responsible for the upregulation of these RAC pathway activators. We probed the promoter regions of the mouse Dock2 and Elmol genes for the consensus SMAD- binding element (SBE) GTCT and the consensus TGFP-inhibitory element (TIE).
  • SBE consensus SMAD- binding element
  • TIE consensus TGFP-inhibitory element
  • the Dock2 promoter contained one TIE located 266bp upstream of the transcriptional start site (TSS) and four SBEs located 207, 497, 559 and 709bp upstream of the TSS (data not shown), and the Elmol promoter contained one TIE located 902bp upstream of the TSS and one SBE located 153bp upstream of the TSS (data not shown). These sites were evolutionarily conserved and not found in repeat regions of the genome. Moreover, these sites were not found in the promoter of Elmo2, despite its high degree of similarity with Elmol .
  • SMAD3 bound to the TIE on the Elmo 1 promoter, but not to the SBE (data not shown), and did not bind to any of the sites identified on the Dock2 promoter (data not shown).
  • Tgfbr2 mRNA was detected by qRT-PCR in YFP+mCherry- cells isolated from tumors generated from Tgfbr2 cKO CD34+ SCC cells infected with full-length Tgfbr2 or YFP+mCherry+ cells isolated from tumors generated from Tgfbr2 cKO CD34+ SCC cells infected with empty vector (data not shown), indicating that mCherry expression faithfully represented cells in which TGFP signaling had been restored.
  • Tgfbr2 in Tgfbr2 cKO CD34+ SCCs dramatically reduced the frequency of mCherry+CD34+ CSCs from 6.5% to 2.5% (data not shown), demonstrating that loss of TGFP signaling is a requirement for CSC maintenance in transition zone carcinoma.
  • rescue of Tgfbr2 abolished Elmol mRNA expression in YFP+mCherry+ cells isolated from tumors generated from Tgfbr2 cKO CD34+ SCC cells infected with full-length Tgfbr2 compared to YFP+mCherry-CD34+ cells isolated from the same tumor (data not shown), confirming that the RAC-activating GEF Elmol is a novel target of TGFP repression.
  • ELMOl Expression of ELMOl is found in human TGFP-deficient invasive anorectal SCC Given that Tgfbr2-deficient anorectal SCC expressed the GEF ELMOl, we investigated whether ELMOl might also be expressed in human anorectal cancers.
  • ELMO 1 was expressed in 5 of the 15 of anorectal tumors tested (data not shown). Concomitant with this expression was a corresponding loss of phosphorylated SMAD2 within the tumor tissue in 5 out of 6 invasive SCC (data not shown).

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Abstract

La présente invention concerne le traitement de carcinomes à cellules squameuses (SCC) et de métastases dérivées de SCC se produisant dans des zones de transition. Selon la présente invention, le SCC est un cancer majeur et reste toujours difficile à traiter en raison de son agressivité et de sa tendance naturelle à développer des métastases. Les inventeurs de la présente invention ont utilisé un modèle de SCC de zone de transition déficiente en Tgfbr2 d'origine murine pour étudier les conséquences de la perte de signalisation de TGFβ dans la propagation tumorale entraînée par des cellules souches cancéreuses et de métastases. Les inventeurs de la présente invention ont découvert un nouveau mécanisme liant la perte de signalisation du TGFβ avec l'invasion et la métastase par l'intermédiaire du GEF ELMO1 activant RAC et ils ont montré que l'inactivation d'ELMO1 altère la métastase du poumon. La présente invention concerne également un agoniste de la voie de signalisation de TGFβ destiné à être utilisé dans le traitement de SCC et/ou pour l'inhibition de l'invasion de carcinomes à cellules squameuses et/ou pour le traitement de métastases issues de SCC chez un sujet en ayant besoin. Les inventeurs de la présente invention ont utilisé particulièrement, un shARN anti-ELMO1.
PCT/EP2018/052308 2017-01-31 2018-01-30 Procédé de traitement de carcinomes à cellules squameuses WO2018141753A1 (fr)

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