WO2018141753A1

WO2018141753A1 - Method for treating squamous cell carcinomas

Info

Publication number: WO2018141753A1
Application number: PCT/EP2018/052308
Authority: WO
Inventors: Géraldine GUASCH; Heather MCCAULEY; Véronique Chevrier
Original assignee: 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
Priority date: 2017-01-31
Filing date: 2018-01-30
Publication date: 2018-08-09

Abstract

The present invention relates to the treatment of squamous cell carcinomas (SCC) and metastasis derived from SCC occurring at transition zones. SCC is a major cancer and is still difficult to treat because of its aggressiveness and its propensity to develop metastasis. Here, the inventors used a murine Tgfbr2 deficient transition zone SCC model to study the consequences of loss of TGFβ signaling in cancer stem cells-driven tumor propagation and metastasis. They uncovered a novel mechanism linking loss of TGFβ signaling with invasion and metastasis via the RAC-activating GEF ELMO1 and they showed that knocking down Elmol impairs metastasis to the lung. Thus, the present invention relates to a agonist of the TGFβ signaling pathway for use in the treatment of 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 SCC in a subject in need thereof. Particularly, the inventors used an shRNA anti-ELMO1.

Description

METHOD FOR TREATING SQUAMOUS CELL CARCINOMAS

FIELD OF THE INVENTION:

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. BACKGROUND OF THE INVENTION:

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]. Populations of cells with 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. As many as 86% of esophageal tumors arise in association with Barrett's esophagus at the esophageal- gastric junction [Trudgill NJ et al, 2003]. 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 (SCC) develop between the stratified epithelium of the anal canal and the simple epithelia of the rectum [Grodsky L., 1961].

To date, we still lack a clear understanding of the signaling and cellular mechanisms that drive transitional epithelial carcinogenesis. Deregulated TGFP signaling seems to be a hallmark of aggressive transition zone cancers. In cervical cancer, 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]. In genital SCC, 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]. However, 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. Within the gastric transition zone, 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]. The backskin of 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 (GTPases) 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) are required to promote the active, GTP-bound form of RAC, and GTPase activating proteins (GAPs) return RAC to its inactive, GDP-bound state [Parri M et al, 2010]. More than 70 GEFs have been described, which 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].

Subsets of long-lived tumor-initiating stem cells or cancer stem cells (CSCs) are often resistant to cancer therapies and thus may be responsible for tumor recurrence. They sustain tumor growth through their ability to self-renew and to generate differentiated progeny, and they may play a role in metastasis. To date, the cellular and molecular mechanisms of Tgfbr2- deficient transition zone carcinoma development and metastasis are unknown.

SUMMARY OF THE INVENTION: 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. Using RN A- Sequencing and chromatin immunoprecipitation, they uncovered a novel mechanism linking loss of TGFP signaling with invasion and metastasis via the RAC-activating GEF ELMO 1. They show that Elmol is a novel target of TGFP signaling via SMAD3 and that restoration of Tgfbr2 results in complete block of ELMOl in vivo. Knocking down Elmol impairs metastasis to the lung, providing a new therapeutic avenue to target the early phase of metastasis in highly aggressive transition zone tumorigenesis.

Thus, 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.

DETAILED DESCRIPTION OF THE INVENTION:

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.

As used herein, the term "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-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.

In a particular embodiment, 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.

Thus, in another word, 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.

In another particular aspect, 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.

In a particular embodiment, 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).

In another particular embodiment, 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.

In another particular embodiment, 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.

In still another particular embodiment, 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.

As used herein, the term "aggressive or highly aggressive squamous cell carcinomas (SCC)" denotes a SCC with an important potential to form metastasis.

As used herein, the term "Tgfbr2" for "transforming growth factor, beta receptor Π" 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.

As used herein the term "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.

As used herein, "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.

Thus, in a particular embodiment, 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.

Thus, in particular embodiment, 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.

As used herein, 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 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 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.]).

In one embodiment, the compound according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). 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.

In one embodiment, the antagonist according to the invention (antagonist of ELMO 1) 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. Various adjuvants known in the art can be used to enhance antibody production. Although 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). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) 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. Alternatively, 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. For the most part, 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. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, 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. In general, 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. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of ELMOl are selected.

In a particular embodiment, the antibody according to the invention may be the ab 174298 sell by Abeam.

In another embodiment, 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. The term "VHH" refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term "complementarity determining region" or "CDR" refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The 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. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is 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. For example, 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).

In one embodiment, 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.

In one embodiment, 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. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. 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).

Then, for this invention, neutralizing aptamers of ELMO 1 are selected.

In one embodiment, the compound according to the invention is a polypeptide.

In a particular embodiment the polypeptide is an antagonist of ELMO 1 and is capable to prevent the function of ELMO 1. Particularly, the polypeptide can be a mutated ELMOl protein or a similar protein without the function of ELMOl .

In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide" to allow the penetration of the polypeptide in the cell.

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 polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, 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.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in 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. In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. 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. In example 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. 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.

Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. 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. In a different approach, 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.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, 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) are linked to the a- and e-amino groups of lysine through stable urethane linkages. 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). In general, 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).

In addition, to the polymer backbone being important in maintaining circulatory half- life, and biodistribution, 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. For example, this type of 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.

In another embodiment, the ELMOl inhibitor according to the invention is an inhibitor of ELMOl gene expression.

Small inhibitory RNAs (siRNAs) 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). 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. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

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.

Both 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. In its broadest sense, 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 . Preferably, 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. In general, 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. One can readily employ other vectors not named but known to the art. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. 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. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which 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. Reportedly, 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. In addition, wild- type 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.

Other vectors include plasmid vectors. 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. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, 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. It may also be administered into the epidermis or a mucosal surface using a gene-gun. 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.

In a particular embodiment, 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 For example, 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.

In one embodiment, 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.

In a particular embodiment, the shRNA use to inhibit the expression of ELMOl may have the sequence CCATCTTATACGACTCAAATT (SEQ ID NO: 1).

In a particular embodiment, 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.

In order to test the functionality of a putative ELMOl antagonist or putative inhibitor of the expression of ELMOl 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, 5xl0⁴ 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. Cells are washed with PBS twice to remove detached cells and incubated with medium containing the inhibitor The cells are observed under an inverted light microscope (Carl Zeiss) equipped with a CCD camera (Ropper) at X10 objective. The wound widths of different area at each time points are measured with MetaMorph software. Data result from calculating the slope of linear trend curve of wound widths as a function of time. In vivo, the use of the antagonist/inhibitor will inhibit cancer cells to move out of the tumor mass, invade the stroma and colonize the lung. 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.

In another embodiment, the invention 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.

In another embodiment, the invention 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.

In another embodiment, another compound used to usually treat squamous cell carcinomas (SCC) 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 . Particularly, Fluorouracil (5-FU), imiquimod or radiotherapy may be used in combination with an agonist of the TGFP signaling pathway.

Therapeutic composition

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.

In a particular embodiment, the invention relates to 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.

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 or subcutaneous administration and the like.

Preferably, 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.

Pharmaceutical 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.

For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide

(Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).

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, 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 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.

In the present methods for treating cancer 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. 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 non- opioid 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. 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: Knockdown of Elmo 1 in vitro affects cell migration.

(A) shRNA knockdown of Elmo 1 in cKO SCC CD34+ cells with two constructs resulted in 40-50% reduction in endogenous Elmol mRNA expression compared to cKO SCC CD34+ cells infected with control shRNA (SH02). Asterisks denote statistical significance using paired-sample Wilcoxon Signed Rank test; p*=0.0350 (Elmol shRNA#l), p*=0.0355 (Elmol shRNA#2). (B) Quantification of the wound healing assay showing the slope of linear trend curve of wound widths normalized to SH02 control. For each construct three different cell lines have been tested and the experiments have been repeated five time. Asterisks denote statistical significance using two-tailed, unpaired student's t-test; p***<0.0001 (Elmol shRNA#2), p***=0.0002 (Elmol shRNA#2 + ELMOl *).

Figure 2: Knockdown of Elmol inhibits Tgfbr2-deficient SCC metastasis.

(A-B) Whole lungs from mice bearing tumors from orthotopic transplantation of Tgfbr2 cKO CD34+ SCC cells infected with SH02 control and Elmol shRNA were dissociated and total YFP+ cells were quantified by FACS (n=6 different mice analyzed for each construct). Per-CP was used to exclude auto-fluorescent cells. Data represent the mean + standard deviation. Asterisks denote statistical significance using two-tailed, unpaired student's t-test; *p=0.0318.

EXAMPLE:

Material & Methods

Mice

The conditional knockout 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 x 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].

Fluorescence-activated cell sorting (FACS)

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). Immediately before sorting, cells were incubated with 7-amino-actinomycin D (7-AAD, eBioscience, 20μ1 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. Cells isolated for tissue culture were collected in epithelial cell culture media(68) (E media) containing 0.05mM calcium, centrifuged at lOOORPM for 5 minutes at 4C, resuspended in E media containing 0.3mM calcium, and 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].

Histological Analysis

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. Alternatively, using a protocol optimized to preserve YFP expression, 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].

Immunostainings and antibodies

Deparaffined tissue sections (5μιη) were subjected to antigen retrieval and immunostaining as previously described. Frozen tissue sections (ΙΟμιη) 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, Danvers, MA, 1/100). 4',6-diamidino-2-phenylindole (DAPI) was utilized as a marker of cell nuclei (Sigma Chemical Co., St. Louis, MO, 1/5,000). Secondary antibodies conjugated to Alexa Fluor 488 or 540 or 649 (Jackson ImmunoResearch, West Grove, PA) were used at a dilution of 1/1,000. For immunohistochemistry, slides were stained with the ABC kit (Vector Laboratories, Burlingame, CA) and counterstained with nuclear fast red (Sigma Chemical Co., St. Louis, MO, USA) according to manufacturers' instructions. Confocal images were acquired by capturing Z-series with 0.3 μιη step size on a Zeiss LSM 880 laser scanning confocal microscope. Images in different focal planes were combined using the Zen software.

Isolation of primary cells and cell culture

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. After expanding MEFs, confluent plates were trypsinized with 0.05% Trypsin-EDTA (Gibco) and irradiated with 60Gy by the CCHMC Comprehensive Cancer Core Facility. Irradiated MEFs were replated at 100% confluency in DMEM containing 10% serum and 1% penicillin- streptomycin one day before sorting and plating tumor cells. On the day of the sort, the media on the irradiated MEFs was changed to E media containing 0.3mM calcium. Clones begin to appear after 7-10 days of culture, and were passaged by transferring individual clones of cells on Whatman paper to a new plate on a feeder layer of irradiated MEFs. After the third passage, 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), β-actin (Sigma, 1/2,000), RAC1 (Cell Signaling, 1/2000), ELMOl (AbCam, 1/2000), a-Tubulin (Sigma, 1/5000), GAPDH (Santa Cruz, 1/5000). 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).

Real-time PCR

Total RNA was isolated using a Qiagen Rneasy Micro Kit and used to produce cDNA using the Maxima first strand cDNA synthesis kit (Fermentas, San Jose, CA). Reverse transcription (RT) reactions were diluted to lOng/μΙ and Ιμΐ 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.

RNA-Seq

All genomic analysis was performed in GeneSpring NGS. Samples were sequenced using the HiSeq 2000 (Illumina, CA) with 50 bp, single-end reads. Following primer and barcode removal, sequences were aligned to the mm9 mouse genome using Ensembl transcripts. Following alignment, reads were quantified to generate computing reads per kilobase per million reads (RPKM), then normalized using the DESeq algorithm and baselining to the median of all samples. We applied a filter, requiring at least 20 reads in at least one of the four samples. We generated a list of differentially regulated genes by comparing CD34-high samples to CD34-negative samples, with a fold change cutoff of 2.0 (n=896 entities). Entities were exported to ToppCluster in order to identify enrichment in previously published microarray datasets (Coexpression Ontologies). A network consisting of genes and associated studies was generated through ToppCluster and Cytoscape. Chromatin Immunoprecipitation

Chromatin immunoprecipitation was performed as previously described. Briefly, 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.

Plasmids and Lentiviral Infection

Using EcoRI and Xhol restriction enzymes, 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) and 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 8μg/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. To confirm the rescue, cells were treated with recombinant human TGFpi (R&D Systems, Minneapolis, MN, USA, 2 ng/ml) for 1 hour at 37°C before cells were trypsinized and processed for RNA extraction for qPCR or lysed for protein extraction and Western blot. Experiments were performed three times in triplicate and statistical significance was determined using paired two-tailed Student's t-test.

shRNA knockdown

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. 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 8μg/ml polybrene (Hexadimethrine bromide, Sigma) and 3ml E Media with 0.05mM Ca++, applied to Tgfbr2 cKO CD34+ SCC cells seeded at 60% confluency 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++. Beginning 48 hours after infection, ^g/ml puromycin was added with fresh E Media with 0.05mM Ca++ every other day to select for infected puromycin-resistant clones.

Mutagenesis Assay

To create a construct that is not recognized by the Elmol shRNA construct #2 (TRCNOOOOl 12656), we created three bases mutations in its target sequence without affecting ELMOl function. The Geneart site-direct mutagenesis kit (Thermo fisher Scientific) was used on pCMV-Sport6 expressing the full-length Mus musculus Elmol gene (2,2Kb) (GE Dharmacon), sequenced and subcloned into the multicloning site of the pLVX-IRES-mcherry lentiviral vector (clonetech, Mountain View, CA) using the NEB Quick Ligation Kit (New England Biolaps, Ipswitch, MA). Lentiviral vectors were produced by SFR Biosciences Gerland Lyon Sud.

Quantification of lung metastasis by FACS

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). Whole lungs were sorted for YFP and analyzed using a FACS Aria II (BD Biosciences) and FACSDiva software (BD Biosciences) in the Research Flow Cytometry Core at CCHMC. Percent YFP+ cells was calculated by dividing number of YFP+ cells by total cells after gating for appropriately sized single cells by forward scatter and side scatter. Experiments were performed twice in triplicate and statistical significance was determined using paired two-tailed Student's t-test.

In vitro wound healing assay

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 μg/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.

Rac Activity Assay

Cell culture dishes at 80 % confluence were washed with ice-cold PBS IX, lysed with 500 μΐ 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. 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. 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. Cell Proliferation Assay

Cells were stained with 10 μΜ 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.

Results

Tgfbr2-deficient anorectal SCC contain a distinct population of cells with clonogenic potential

Mice lacking Tgfbr2 in stratified epithelia expressing Keratin 14 (K14) develop spontaneous squamous cell carcinoma (SCC) at the transition zone between the anal canal and rectum [Guasch G et al, 2007]. To lineage trace Tgfbr2 deficient cells within these transitional SCC and enable isolation of specific Tgfbr2 deficient tumor cell populations, we backcrossed these mice into mice containing loxP sites flanking a STOP sequence preceding eYFP inserted into the Rosa26 locus (data not shown), such that all K14-positive epithelial cells, including the anorectal SCC cells, while conditionally null for Tgfbr2 expressed YFP (cKO mice, data not shown). We had previously identified a population of cells with stem cell characteristics, including colocalization with known stem cell markers, such as CD34, in the anorectal transition zone of wild-type mice [Runck LA, et al, 2010]. We hypothesized that tumors arising at the anorectal transition zone in the Tgfbr2 cKO mice would contain a population of CD34- expressing cells, and that these cells would represent a population of tumor-propagating cells or so-called cancer stem cells (CSCs). Based on the idea that CSCs should reside at the tumor- stroma border, we thought that CSCs of anorectal SCCs should express abundant integrins. To test this hypothesis, we first analyzed marker expression within histologic sections of Tgfbr2- deficient anorectal tumors. Immunofluorescence staining revealed that all YFP+SCC cells located at the tumor-stroma interface expressed high levels of the hemidesmosomal a6 integrin (data not shown) and the focal adhesion marker βΐ integrin (data not shown), and a fraction of these expressed CD34 (data not shown). These three markers were used to isolate discrete populations of cells by fluorescence-activated cell sorting (FACS). Tumors were dissociated into a single-cell suspension as previously described, stained with antibodies, and subjected to flow cytometry. Blood cells, endothelial cells, macrophages and dead cells were excluded, and live, K14+YFP+ epithelial cells were further purified upon a6-integrin and βΐ -integrin expression. Of these live, YFP+, a6-integrin+, pi-integrin+ cells, distinct CD34- and CD34+ populations of cells were observed and isolated (data not shown). The frequency of epithelial CD34+ cells within the tumor varied between mice, from 7% to 34%. When plated on a feeder layer of irradiated fibroblasts, 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). These data suggest that the CD34+ population of epithelial cells isolated from the Tgfbr2 cKO anorectal SCC has self- renewal properties in vitro.

Tgfbr2 cKO CD34+ SCC cells are enriched for in vivo tumorigenicity

We next sought to determine whether the CD34+ SCC cells were enriched for self- renewal properties and tumorigenicity in vivo. We previously developed an orthotopic transplantation assay in which anorectal SCC cells are injected specifically and reproducibly within the anorectal transition zone of immunocompromised Nu/Nu mice. Orthotopic injection of 200-106 CD34+ cells from Tgfbr2 cKO anorectal SCC into recipient Nu/Nu mice caused secondary tumor formation with 100% efficiency (n=89) (data not shown). These secondary anorectal tumors were invasive, moderately- to poorly-differentiated SCC, characterized by cellular atypia, squamous nests with keratin pearls, intercellular bridges, aberrant mitoses, cellular disorganization and desmoplastic stroma (data not shown), and were morphologically similar to the primary Tgfbr2 cKO tumors of origin. Just as in the primary Tgfbr2 cKO SCC, a population of YFP+ epithelial cells expressing CD34 could be identified by immunofluorescence staining (data not shown) and isolated by FACS using the same strategy as used previously (data not shown). 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). We confirmed that loss of Tgfbr2 was maintained in YFP+ SCC cells by qPCR (data not shown) and immunofluorescence staining, whereas TGFpRII and nuclear pSMAD2 were still present in the surrounding stroma (data not shown).

To determine whether SCC CD34+ cells were tumorigenic, CD34+ and CD34- YFP+ epithelial cells were isolated from the secondary anorectal tumor by FACS and orthotopically transplanted into the anorectal transition zone of Nu/Nu mice. After tertiary transplant of CD34+ SCC cells, 8/13 mice developed anorectal tumors, compared to 1 mouse of 13 transplanted with CD34- SCC cells (data not shown). YFP+ cells negative for a6-integrin and βΐ-integrin were unable to form tumors upon transplantation (n=14), indicating that not every cell type within the Tgfbr2 cKO anorectal SCC is tumorigenic. 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). In fact, in the single tertiary tumor that formed after transplant of CD34- SCC cells, 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. Whereas CSC marker expression may be dynamic, CD34 remains a useful marker to assay the CSC properties of a population of cells isolated from murine SCC. Taken together, the ability of 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

Squamous cell carcinomas, including those occurring at transition zones, frequently metastasize to the lung. We analyzed the lungs of tumor-bearing mice and observed that 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. Furthermore, Tgfbr2 cKO lung metastases expressed YFP and contained populations of CD34+ cells (data not shown), recapitulating the hierarchy of the primary tumor of origin. Sequencing of R A from CD34+ and CD34- Tgfbr2 cKO SCC cells, isolated by FACS, revealed that CD34+ SCC cells were enriched for an invasive and metastatic gene signature as well as for mRNA involved in the RAC/RHO/RAS pathway (data not shown). Using ToppCluster to generate a network of genes shared between Tgfbr2 deficient CD34+ anorectal transitional SCC cells and published datasets of aggressive human cancers (data not shown) we found that a number of genes were commonly overexpressed in human cancers including cervical carcinoma. We validated a selection of the most upregulated genes in the CD34+ SCC cells by qPCR (data not shown), including Cathepsin S, Fibrillinl, Sppl , Mmp9 and Tgf]32, which are all implicated in ECM organization, invasion and metastasis, and members of the RHO GTPase pathway Rac2, RhoH, RhoJ, Vavl , Dock2, and Elmol . We confirmed that the GEF ELMOl is co-expressed with CD34-positive tumor epithelial cells at the protein level by immunofluorescence staining (data not shown) and observed increased staining of this RAC-activating factor at the leading edge of the tumor. We also confirmed that RAC2 is co-expressed with CD34-positive cells (data not shown) and that RAC1 is strongly expressed at the tumor-stroma border of Tgfbr2 cKO SCC (data not shown). A strong RAC activity was confirmed in vitro when we analyzed the amount of GTP-bound RAC in the Tgfbr2 cKO SCC CD34+ cell line (data not shown). These results indicate that, while Racl mR A was not found to be upregulated in CD34+ cells by RNA-Seq analysis, RACl protein activity may be elevated in CSCs by upregulation of GEFs. Taken together, these data implicate CD34+ CSCs in metastasis of Tgfbr2 deficient SCC, potentially through upregulation of the RHO/RAC GTPase pathway.

Upregulation of RAC signaling is unique to the anorectal Tgfbr2 cKO SCC and not found in Hras-induced skin SCC

We hypothesized that aberrant RAC signaling was, at least in part, responsible for the highly aggressive nature of transition zone cancers. We compared the profile of CD34+ CSCs from our Tgfbr2 cKO anorectal SCC to the CSCs purified from malignant skin SCC in mice from various genetic backgrounds treated with the chemical mutagen 7,12-dimethyl-benz[a] anthracene (DMBA) to induce an activating mutation in Hras. In DMBA-induced Tgfbr2 cKO skin SCC, Schober et al reported two CSC populations: CD34Hi and CD34Lo, with the CD34Lo CSC population demonstrating increased tumorigenicity over the CD34Hi population. Bioinformatics analysis revealed that only 6 genes were common between the CSC from Tgfbr2 cKO anorectal CD34+ cells and DMBA-induced Tgf r2 cKO skin CD34Hipopulation (***p value=l .57x10-7) and 7 genes were common in the DMBA-induced Tgfbr2 cKO skin CD34Lopopulation (***p value=7.48xl0-5) (data not shown). As expected in malignant cancers, these genes are involved in ECM organization, epithelial to mesenchymal transition (EMT) and metastasis but none was related to the RAC/RHO/RAS pathway. When we compared the skin CSC population from DMBA-induced Tgfbr2 and focal adhesion kinase (FAK) double KO mice, in which SCC initiation is delayed compared to Tgfbr2 cKO skin, 9 genes were common between the Tgfbr2 cKO anorectal CD34+ CSCs and the CD34Lopopulation (***p value=l .98x10-10). Similarly, only 11 genes involved in ECM organization and other functions were common between the Tgfbr2 cKO anorectal CD34+ CSCs and the DMBA-induced skin CD34Lopopulation in genetically wild-type mice (***p value= 1.49x10-11). The analysis of skin SCC signatures from FAK single KO mice, which are more refractory to DMBA-induced SCC formation, showed 21 common genes between Tgfbr2 cKO anorectal CD34+ CSCs and the CD34Lopopulation (***p value=5.35xl0-26) and one gene related to the RAC/RHO/RAS pathway was found in common (Pdgfrb). We also compared our Tgfbr2 cKO anorectal SCC CD34+ CSC gene signature with DMBA-induced skin SCC CD34+ cells with overexpression of VEGF, which accelerates tumor growth. We found 40 genes that are upregulated in Tgfbr2-deficient anorectal CD34+ CSCs yet downregulated by VEGF in comparison to DMBA-induced backskin CD34+ CSCs (***p value=2.69x10-53), and we found 45 genes commonly upregulated between Tgfbr2-deficient anorectal CD34+ CSCs and DMBA-induced SCC CD34+ skin CSCs when mice overexpressed VEGF (***p value=2.62x10-39). Among these data sets, we found overlap of three RAC/RHO/RAS family genes with Tgf r2-deficient anorectal CD34+ CSCs, suggesting that dysregulation of the RAC/RHO/RAS pathway may be associated with CSCs within highly aggressive tumors.

Immunofluorescence staining confirmed that TGFP-deficient DMBA-induced skin SCC does not express RACl and RAC2 in contrast to anorectal SCC, where these proteins are found at the invasive front of the tumor (data not shown). These analyses suggest that aberrant RAC signaling may be a hallmark of highly aggressive tumors arising spontaneously in tumor-prone transition zones.

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 wondered 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). 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 . We used chromatin immunoprecipitation (ChIP) to determine whether SMAD3, a canonical effector of TGFP signaling, bound any of these potential SBEs or TIEs. 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). These results indicated that Elmol is a previously unidentified direct target of TGFP signaling via SMAD3.

Restoration of TGFpRII results in complete block of ELMO 1 in vivo

Because we identified a direct link between SMAD3 and the promoter region of Elmol, we hypothesized that rescue of TGFpRII in Tgfbr2 deficient cells would reduce ELMOl expression. We cloned the full-length Tgfbr2 gene into the pLVX-IRES-mCherry lentiviral vector and infected Tgfbr2 cKO CD34+ SCC cells with this construct or the empty vector. Infection of YFP+CD34+ SCC cells with the rescue construct, but not the empty vector, efficiently restored Tgfbr2 mRNA expression (data not shown). Rescued CD34+ SCC cells became sensitive to TGFP treatment and phosphorylated SMAD2 (data not shown). Orthotopic transplantation of rescued CD34+ SCC cells resulted in a two-fold delay in tumor latency compared to Tgfbr2 cKO CD34+ SCC cells infected with the empty vector (data not shown), although all mice eventually developed tumors due to the inefficient infection rate of the rescue vector (data not shown). Tumors generated from the orthotopic transplantation of Tgfbr2 cKO CD34+ SCC cells infected with the empty vector or rescued with full-length Tgfbr2 were dissociated and YFP+mCherry+, YFP+mCherry-CD34+ and YFP+mCherry-CD34- cells were isolated by FACS (data not shown) and subjected to RNA extraction. YFP+mCherry+ cells isolated from tumors generated from rescued Tgfbr2 cKO CD34+ SCC cells infected with full- length Tgfbr2 expressed Tgfbr2 mRNA 250-fold over YFP+mCherry- cells isolated from the same tumor or YFP+mCherry+ cells isolated from tumors generated from Tgfbr2 cKO CD34+ SCC cells infected with empty vector (data not shown). No 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. Restoration of 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. Importantly, 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.

Expression of ELMOl is found in human TGFP-deficient invasive anorectal SCC Given that Tgfbr2-deficient anorectal SCC expressed the GEF ELMOl, we wondered whether ELMOl might also be expressed in human anorectal cancers. We performed immunohistochemistry on a series of human anorectal biopsies that ranged from normal mucosa to invasive grade 3 carcinomas. We found that 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). Interestingly, none of the early stage tumors tested (anal intraepithelial neoplasia or SCC in situ) expressed ELMOl, and these specimens stained strongly for nuclear phosphorylated SMAD2 similarly to normal anorectal mucosa (data not shown). Taken together, these data support a role for ELMOl in invasive TGFP-deficient transition zone SCC.

Knockdown of Elmol diminishes cell migration in vitro, RAC localization at the tumor- stroma border, and reduces markers of invasion in Tgfbr2-deficient SCC CD34+ CSCs

To determine whether a reduction in ELMOl could reduce invasion and metastasis in Tgf r2 cKO SCC, we knocked down Elmol in Tgf r2 cKO CD34+ SCC cells using two shR A constructs (Figure 1 ). Treatment of Tgfbr2 cKO CD34+ SCC cells with Elmo 1 shRNA resulted in 40% (construct #1) or 50%> (construct #2) reduction in endogenous Elmol mRNA levels, compared to cells infected with control shRNA (SH02) (Figure 1A). Western blot analysis confirms this reduction at the protein level (data not shown). To confirm the specificity of the shRNA, we used a hairpin-resistant ELMOl cDNA (ELMOl *) in which we had introduced three base mutations in the target sequence of the Elmol shRNA #2 without affecting the function of ELMOl (data not shown). We cloned this construct into the pLVX- IRES-mCherry lentiviral vector and infected Tgf r2 cKO CD34+ SCC Elmol shRNA expressing cells with ELMOl * or the empty vector. Western blot analysis confirmed that overexpression of the hairpin-resistant ELMOl * construct restores similar level of expression of ELMOl (data not shown). We performed an in vitro wound healing assay to show that knocking down Elmol in Tgfbr2 cKO CD34+ SCC cells affected their ability to migrate and close the wound. This effect was rescued when Elmol knockdown cells expressed the hairpin- resistant ELMOl * construct (Figure IB). We confirmed that the effect in cell migration was not due to a difference in proliferation as measured by flow cytometry (data not shown).

We transplanted these cells orthotopically into the anorectal transition zone of recipient mice and observed tumor formation with no difference in latency. Consistent with this result, we observed that Elmol knockdown does not affect the proportion of CSC CD34+YFP+mCherry+ cells analyzed by FACS (Control SH02: 4.1% compared to Elmo l shRNA: 3.3%, data not shown) We isolated epithelial YFP+ CD34+ cells from these tumors, using the same FACS strategy already described, and observed a 60% reduction in Elmo l mRNA compared to CD34+ cells isolated from SH02 tumors, validating in vivo the loss of Elmol (data not shown). Infection of the cells with the pLVX-mCherry vector did not affect the efficiency of the shRNA, as we observed a similar reduction in Elmol mRNA compared to CD34+ cells isolated from SH02 tumors that did not express the mCherry (50% reduction for construct #1 and 60%> reduction for construct #2 (data not shown). Using immunofluorescence we confirmed reduction in ELMOl expression at the protein level in these tumors (data not shown) compared to SH02 tumors. Infection of CD34+ Elmol shRNA SCC cells with the hairpin-resistant ELMOl * construct efficiently restored Elmol mRNA and ELMOl protein expression in the resulting tumors (data not shown), indicating that mCherry expression faithfully represented cells in which ELMOl expression had been restored. Consistent with a migration defect in vitro, we observed a dramatic reduction in RACl staining at the tumor- stroma border in tumors with Elmol knockdown (data not shown), which was rescued in cells expressing the hairpin-resistant ELMOl * construct (data not shown).

Because RAC signaling plays a role in tumor invasion, we hypothesized that a number of markers of EMT and/or invasion would be altered upon Elmol knockdown, and indeed observed reduction in the mRNA expression of Snail, aSma, Vimentin, Zeb2 and Mmp9 in CD34+ cells isolated from Tgfbr2 cKO tumors infected with both Elmol shRNA constructs #1 and #2 compared to those infected with SH02 control (data not shown). The level of mRNA expression of these genes was restored or increased in the cells expressing the hairpin-resistant ELMOl * construct (data not shown). This increase can be explained by the fact that the level of Elmol in the rescued cells was higher than endogenous levels in the SH02 control cells. These results indicate that Elmol reduction alters localization of RAC in Tgfbr2-deficient SCC in vivo and reduces the expression of invasive markers in CD34+ CSCs.

Knockdown of ELMOl inhibits metastasis in Tgfbr2-deficient SCC

To determine whether Elmol knockdown altered tumor progression and metastasis in Tgfbr2 cKO SCC, we screened the lungs of tumor-bearing mice for YFP+ metastases by FACS. We observed a dramatic overall reduction in the number of YFP+ cells in the lungs of mice orthotopically transplanted with Tgfbr2 cKO CD34+ cells infected with Elmol shRNA knockdown compared to those infected with SH02 control (n=6 mice in each condition) (Figure 2 A and B). We detected 1.4% YFP+ cells in the lungs of mice transplanted with the SH02 control cells, whereas we detected a dramatic reduction to 0.4% YFP+ cells in mice transplanted with the ELMOl shRNA#l and failed to detect any YFP+ cells in the lungs of mice transplanted with ELMOl shRNA #2. We serially sectioned and stained the whole lungs from additional tumor-bearing mice, and observed YFP+ metastatic nodules in mice transplanted with SH02 cells, but never in mice transplanted with Elmo l shR A-infected cells (data not shown), validating the sensitivity of FACS in providing a quantitative method to screen for lung metastases. Taken together, these data demonstrate that upregulation of the GEF ELMOl is required for Tgfbr2 deficient SCC CD34+ CSCs to metastasize.

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Claims

CLAIMS:

1. 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.

2. An agonist of the TGFP signaling pathway according to claim 1 wherein said agonist is an agonist of Tgfbr2 or an antagonist of ELMO 1 or an inhibitor of the expression of ELMOl .

3. An antagonist of ELMOl or an inhibitor of the expression of ELMOl according to claim 2 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.

4. An inhibitor of the expression of ELMOl according to claim 3 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.

5. An inhibitor of the expression of ELMOl for use according to claim 4 wherein the inhibitor of the expression of ELMOl is a shRNA and has at least 80% of homology with the sequence SEQ ID NO: 1.

6. An agonist for use according to claim 1 wherein another compound selected from the group consisting in Fluorouracil (5-FU) and imiquimod is used in combination with said agonist.

7. A therapeutic composition comprising 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.