CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/650,464, filed May 22, 2012, and U.S. Provisional Patent Application No. 61/620,249, filed April 4, 2012, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant R21 awarded by the United States National Institutes of Health and National Institute of Dental and Craniofacial Research. The government has certain rights in the invention.

INTRODUCTION

[0003] The progression from a locally growing tumor to an invasive and metastatic tumor is the event that is most often responsible for treatment failures in patients with cancer. Head and neck squamous cell carcinoma (HNSCC) is highly aggressive and accounts for 45,000 malignancies diagnosed each year and is the fifth leading cancer worldwide. Despite various treatment options, HNSCC patients are still faced with a high chance of recurrence and/or metastasis, which is responsible for the poor clinical prognosis, with a 5-year survival rate of only about 50 percent.

[0004] Metastatic dissemination requires several steps including loss of cell-cell adhesions and cell polarity, acquisition of a motile phenotype, delamination of individual or small collective groups of cells from the primary tumor, subsequent reorganization of the extracellular matrix (ECM), and cell migration to adjacent and distant tissues. During this process of epithelial-to- mesenchymal transition (EMT), cells downregulate their epithelial-specific tight and adherens junction proteins like E-cadherin and cytokeratins and re-express mesenchymal molecules like vimentin and N-cadherin. Various factors play a crucial role in malignant tumor progression, such as altered signal transduction pathways, changes in adhesive and migratory capabilities of tumor cells, and the tumor microenvironment. At this stage of tumor development, tumor cells migrate into and invade the surrounding tissue, thereby forming an invasive front enriched in integrin-mediated interaction of the tumor cells with the ECM. [0005] Metastatic seeding leads to most of the morbidity from carcinomas. Because of the complex nature of tumor metastasis, our current understanding of the process remains very descriptive. The most challenging questions in metastasis are to understand the rate-limiting steps during metastasis and to define the genetic and epigenetic changes conferring such behaviors. EMT has been proposed to describe the dynamic changes like loss of cell-cell adhesion and increased motility that carcinoma cells experience to achieve local invasion and dissemination to distant organs. However, it remains a challenge to observe EMT in human carcinomas. One major difficulty is caused by the transient, reversible nature of EMT during carcinoma invasion and metastasis. Because only a small minority of carcinoma cells may be invasive and undergo an EMT in primary tumors, the alteration of gene expression in such cells can be masked by the bulk of non-metastatic cells. Detecting such transient cells may be useful to assess the contribution of EMT to the behavior of high grade carcinomas and develop novel targets for anti-metastasis therapeutics. Despite the progress gained in recent years, the biomolecular processes involved in cancer metastasis are not clearly understood. Improved methods for preventing, inhibiting, and diagnosing cancer metastasis are desired, as well as experimental metastasis models that can recapitulate the physiologic and pathologic conditions observed in human cancer patients.

SUMMARY

[0006] In an aspect, provided are methods of detecting metastatic cancer in a subject. The method may include measuring an amount of catulin in a sample from the subject, and comparing the amount of catulin in the sample to an amount of catulin in a control, wherein an increased level of catulin in the sample relative to the amount in the control indicates metastatic cancer in the sample. The cancer may comprise squamous cell carcinoma. The cancer may comprise breast cancer. The amount of catulin in the sample may be measured by catulin antigen-antibody binding.

[0007] In another aspect, provided are methods of treating metastatic cancer. The method may include administering to a subject an effective amount of a therapeutic composition adapted to reduce the amount or the activity of catulin. The cancer may comprise squamous cell carcinoma. The cancer may comprise breast cancer. The therapeutic composition may comprise an siRNA adapted to reduce the expression of catulin. The therapeutic composition may reduce or inhibit tumor metastasis. [0008] In a further aspect, provided are methods of determining a perimeter of a metastatic tumor. The method may include obtaining a sample of the tumor or surrounding tissue, measuring an amount of catulin in the sample, and comparing the amount of catulin in the sample to an amount of catulin in a control, wherein an increased amount of catulin in the sample relative to the amount in the control indicates the sample is part of the tumor, and wherein a substantially equivalent amount of catulin in the sample relative to the amount in the control indicates the sample is part of non-tumor tissue beyond the perimeter of the tumor. The cancer may comprise squamous cell carcinoma. The cancer may comprise breast cancer. The amount of catulin may be measured by catulin antigen-antibody binding.

[0009] In a further aspect, provided are methods for monitoring progression of cancer in a subject undergoing therapeutic treatment. The method may include measuring an amount of catulin in a first and a second sample taken from the subject at a first and a second time, and comparing the first and second amounts, wherein an increase in the amount of catulin in the second sample relative to the amount in the first sample indicates a progression of the cancer. The cancer may comprise squamous cell carcinoma. The cancer may comprise breast cancer. The amount of catulin may be measured by catulin antigen-antibody binding.

[0010] In a further aspect, provided are methods for determining the aggressiveness of post-operative cancer therapy for a subject. The method may include obtaining a sample of a tumor from the subject, measuring an amount of catulin in the sample, and treating the subject post-operatively, wherein the aggressiveness of post-operative treatment is dependent on the amount of catulin measured in the sample. The cancer may comprise squamous cell carcinoma. The cancer may comprise breast cancer. The amount of catulin may be measured by catulin antigen-antibody binding.

[0011] In a further aspect, provided is a reporter system for metastatic cancer. The system may include a recombinant cell. The recombinant cell may have a genome, wherein the genome comprises a first polynucleotide corresponding to the 5'-flanking sequence located from about 1.3 kb upstream and to about 200 bp downstream of the human a-catulin gene transcription start site, the first polynucleotide upstream of and driving the expression of a second polynucleotide encoding a fluorescent polypeptide. The recombinant cell may originate from a human SCC cell line or a breast cancer cell line. genome comprises a first polynucleotide corresponding to the 5 -flanking sequence located from about 1.3 kb upstream and to about 200 bp downstream of the human ocatulin gene transcription start site, the first polynucleotide upstream of and driving the expression of a second polynucleotide encoding a fluorescent polypeptidein. The recombinant cell may be grown in a medium. The methods may further include contacting the recombinant cell with the compound, measuring at least one of growth and invasion of the recombinant cell into the medium, comparing the at least one of growth and invasion with a control, and determining the antimetastasis activity of the compound. A reduction in the at least one of growth and invasion relative to the control may indicate antimetastasis activity of the compound.

[0013] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013A] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Figure 1. Catulin is expressed in motile, mesenchymal ceils. (A) Catulin is upreguiated in motile a-catenin KO ceils compared to WT cells. (B and C) RNA isolated from FACS sorted GFP+ SCC tumor and metastatic ceils was used to perform microarray. Arrow shows lymphatic vessel, arrowhead shows metastatic ceils that were dissected for analysis. (D) RT-PCR on independent samples confirmed the upregulation of catulin seen in the microarray data. (E and F) Epithelial SCC cells were induced to go through EMT using various factors as indicated, for 44 hours (20 ng/mL TGF-βΙ , 20 ng/mL EGF, Wnt3a conditioned media in DMEM with 0.1 % FBS). Cellular morphology (£) shows successful EMT induction, which was confirmed by RT-PCR (F) using mesenchymal markers Vimentin and Snail. Catulin expression is upreguiated in the mesenchymal cells.

[0015] Figure 2. Catulin is expressed at the leading edges and in migrating cells in SCCs. (A-F) in a tissue array with 28 cases of human HNSCCs and normal oral mucosa epithelium, representative SCCs are shown (S-F). There is minimal catulin expression in the normal epithelium (A). Tissue origin and tumor grade, as provided by the manufacturer, is indicated. Selected regions from higher grade SCCs (D-F) are magnified (D'-F ^! ). (G-/) Mice expressing β- galactosidase under endogenous catulin promoter were given drinking water containing 4NQO for 16 weeks. Mice were examined 22 and 28 weeks after initial 4NQO administration. 4NQO- induced SCC lesions formed on the tongues compared to untreated controls, with higher grade dysplasia at 28 weeks, reflecting the carcinogenic progression. Staining tongue sections with x- gal, we see catulin expressed at the leading edge of invading sees. ep=epithelium, s=stroma, m=muscle.

[0016] Figure 3. Ablation of catulin in human SCC cell lines decrease their migration and invasion in vitro. (A) shRNA lentiviral system was used to generate stable hSCC cell lines that were catulin-deficient. Stable cell lines transduced with the lentivirus express turboGFP for cell tracking. (S and C) The specific knockdown of catulin in USC-HN1 cells (G85) compared to the non-silencing control (GNS) was confirmed by RT-PCR (B) and western blot (C). HEK293 cells transiently transfected with human catulin-Myc were used as controls. (D and EE) In vitro cell migration (D) and invasion (EE) was decreased in catulin-ablated SCC-15 cells; cells were visualized with toluidine blue. Representative experiments are shown.

[0017] Figure 4. Ablation of catulin in hSCC cell lines decrease their metastatic potential in xenotransplants in vivo. (A) Upon injecting hSCC USC-HN1 non-silencing control cells and those with stable knockdown of catulin into NOD.Cg mice, tumors of similar sizes formed. (S) Cell growth assay showed that knockdown of catulin in these USC-HN1 cells did not affect growth rate. (C-L) Tumors formed from WT control cells (top panel) and catulin knockdown cells (bottom panel) show dramatic decrease in cells that metastasized to the lungs, visualized by tGFP expression. Arrows show metastatic groups of cells. (M) On average, when catulin was ablated, there was about half the number of metastatic cells per lung compared to the WT control.

[0018] Figure 5. Catulin-ablated hSCC cells are unable to rearrange ECM components to migrate and invade in vivo. We analyzed the tumors that formed from the injection of catulin- deficient and WT control hSCC cell lines, where tGFP expression marks the transduced cancer cells. (A and B) H&E staining for general tumor morphology. (C-J) IF staining of the tumors that formed from the injection of catulin-deficient and WT control hSCC cell lines with antibodies as indicated. (K and L) IHC using phospho-JNK specific antibody. Arrows indicate invasive tumor fronts, whereas dashed lines indicate less invasive tumor margins in catulin-deficient tumors.

[0019] Figure 6. Metastatic cells from the lymphatic vessels show mesenchymal morphology after isolation. 3 days after isolation, metastatic hSCC USC-HN1 GFP-labeled cells from the lymphatic vessels (A) show a mesenchymal morphology, compared to USC-HN1 cells from the primary SCC tumor (C). 10 days after isolation, the metastatic cells from the lymphatic vessels (B) revert to an epithelial morphology, typical of USC-HN1 cells, as compared to the cells from the primary SCC tumor (D).

[0020] Figure 7. Ablation of a-catulin in human breast cancer cell line MDA-MB-231 decreases their migration and invasion in vitro. shRNA lentiviral system was used to generate stable MDA-MB-231 cell lines that were a-catulin-deficient. The specific knockdown of a-catulin in MDA-MB-231 cells (G85) compared to the non-silencing control (GNS) was confirmed by RT- PCR (A), real-time qPCR (Α'), and western blot (B). In vitro cell migration (C) and invasion (D) was decreased in a-catulin-ablated MDA-MB- 231 cells; cells were visualized with toluidine blue. Standard error bars are shown {n=4 for cell migration and n=4 for invasion assay). *** statistical significance was determined using f-test (p < 0.001) or * analysis of variant (ANOVA) (p < 0.02).

[0021] Figure 8. Ablation of a-catulin in human squamous cell carcinoma cell line USC- HN1 has no effect on cell growth rate or apoptosis. (A) performing a cell growth assay, it was observed that the knockdown of α-catulin does not affect cell growth in USC-HN1 cells. (B) AnnexinV analysis was performed to look at cell apoptosis. Cells were either left untreated or treated with TNF-a (10 ng/mL) and CHX (10 pg/mL) for 30 hours to induce apoptosis. There was no considerable difference in apoptosis between the control and a-catulin knockdown USC- HN1 cells, either untreated or treated with TNF-a and CHX. One representative experimental result is shown.

[0022] Figure 9. Catulin-GFP positive cells are present in small foci of the tumor. 3 different hSCC (SCC15, SCC25, and USC-HN1 ) cell lines stably expressing a Catulin-GFP reporter, were injected to NOD.Cg mice. After 5 weeks, the primary tumors were dissected and visualized in bright field (A) and with fluorescence (B). Arrow shows the GFP positive areas, dashed lines indicate tumor margin. A tumor formed from the injection of the Catulin- GFP SCC15 cell line is shown as a representative tumor.

[0023] Figure 10. Expression of GFP in our catulin-GFP reporter system correlates with expression of endogenous a-catulin in migratory streams of cancer cells. Tumors that formed from the injection of 3 different hSCC (SCC15, SCC25, and USC-HN1) cell lines stably expressing the Catulin-GFP reporter were analyzed after 5 weeks from initial injection. (A-C) immunofluorescent staining of frozen sections with antibodies as indicated. Arrows indicate invasive tumor fronts in tumors, whereas dashed lines indicate less invasive tumor margins [0024] Figure 11. Catulin-GFP positive cells localize at the invasion front and in the migratory streams of cancer cells. Tumors that formed from the injection of 3 different hSCC (SCC15, SCC25, and USC-HN1) cell lines stably expressing Catulin-GFP reporter were analyzed after 5 weeks from initial injection. (A-C) and (D-F), 2 different examples of immunofluorescent staining of frozen sections with antibodies as indicated. Arrows indicate GFP positive invasive cells (B and E) in the tumor where integrin beta4 staining no longer sharply separates the tumor from stroma (C and F).

[0025] Figure 12. α-catulin is expressed at the invasion front and in migrating cells in SCCs. (A-F), immunohistochemical analyses shows α-catulin expression in a tissue array consisting of 20 cases of human HNSCCs and 6 normal oral mucosa epithelium. Tissue origin and tumor grade, as provided by the manufacturer, is indicated. (A-F), representative images are shown. Asterisks point out the migrating, metastatic tumor cells. There is minimal a-catulin expression in the normal epithelium (A), when compared to the HNSCCs (B-F). (D'-F'), selected regions from higher grade SCCs are magnified.

[0026] Figure 13. Initial sorting of Catulin- GFP+ invasive cells (P8) on the basis of GFP and integrin alpha6 expression. (A) Unstained sample control. (B) Sorted tumor samples.

[0027] Figure 14. Ablation of α-catulin in hSCC cell lines decreases their metastatic potential in xenotransplants in vivo. Upon injecting hSCC USC-HN1 TNS (A) and USC-HN1 GNS (M) (non-silencing control cells) and hSCC USC-HN1 T84 and USC-HN1 G85 (a-catulin knockdown cells) into NOD.Cg mice, tumors of similar sizes formed. The specific knockdown of α-catulin in hSCC USC-HN1 T84 and G85 cells compared to the non-silencing control hSCC USC-HN1 TNS and GNS cells, was confirmed by RT-PCR (B and N), real-time qPCR (Β' and N'), and western blot (C and O). (E-L) and (Q-X), tumors formed from control cells (top panel) and α-catulin knockdown cells (bottom panel) show a dramatic decrease in cells that metastasized to the lungs, as visualized by tRFP (E -L) and tGFP (Q-X) expression, in a-catulin- deficient xenografts. Arrows show metastatic groups of cells. D and P, quantification of metastatic foci formed in the lungs from the injection of control and a-catulin-deficient USC-HN1 tumor cells. Standard error bars are shown. (n=3 for TNS/T84 lentiviral system and n=5 for GNS/G85 lentiviral system). Statistical significance was assessed by t-test (p<0.001).

[0028] Figure 15. 3D system of invasion of hSCC cells into a collagen-rich matrix, (a) Inducible shRNA lentiviral system was used to generate stable hSCC cell lines that were catulin-deficient upon doxycycline induction, (b) Stable cell lines transduced with the lentivirus express turboRFP for cell tracking, (c and d) The specific knockdown of catulin in hSCC USC- HN1 cells (T84) was compared to the non-silencing control (TNS) after introducing doxycycline for 72 hrs and was confirmed by RT-PCR (c) and western blot (d). (A and D) Catulin control and catulin KD SCC cells were cultured in sphere inducing media and transduced with baculovirus to introduce actin-GFP expression. Spheres were then placed into the mixture of collagen and matrigel and after 48 hours confocal microscopy was performed (B, C-C") catulin control, (E, F- F') catulin KD). Note invasive cells marked by arrows in (B and C) in catulin control tumor cells and lack of invasion into the matrix of catulin deficient tumor cells marked by asterix (E and F).

[0029] Figure 16. Ablation of a-catulin in hSCC cell lines have decreased invasiveness in vivo. We further analyzed the tumors that formed from the injection of control and a-catulin deficient USC-HN1 cell lines. We can observe and track injected cells by the tGFP reporter. A and B, checking for proliferation rate, we do not see a considerable difference between the two tumors, as assessed by the proliferative marker, Ki67. C and D, when analyzing the tumors for human-specific lymphatic vessel marker, lyve-1 , the control tumor (C) had collective groups of cells that were able to invade the surrounding stroma and form their own lymphatic vessels, whereas a-catulin-deficient tumors (D) were unable to do so. (E-F), IHC staining using phospho- JNK antibody show an increase in activated phospho-JNK in control tumor (E) compared to a- catulindeficient tumor (F). Arrows indicate invasive tumor fronts in control tumors, whereas dashed lines indicate a less invasive tumor margin in a-catulin-deficient tumors.

DETAILED DESCRIPTION

[0030] Among the many changes in gene expression and protein function that occur during tumor progression, alterations in cell-cell and cell-matrix adhesion seem to have a central role in facilitating tumor cell migration, invasion, and metastatic dissemination. E-cadherin and/or a- catenin are lost concomitantly with tumor progression in most epithelial cancers. Reduced E- cadherin and a-catenin are often prognostic markers of poor clinical outcome in squamous cell carcinomas (SCCs). In a mouse model of SCC, the conditional loss of cell-cell junction protein a-catenin in the epithelium resulted in the formation of SCC-like phenotype, accompanied by increased cell proliferation and migration. Microarray analysis performed using α-catenin knockout (KO) cells that failed to form cell-cell junctions and had increased proliferation and motility, and WT epithelial cells, showed an upregulation of a new α-catenin homologue, a-catenin-like 1 , catulin. The catulin protein serves as a scaffold for Rho signaling and shows structural similarities to vinculin and a-catenin, particularly in the N-terminal region, which contains binding sites for β-catenin, talin, a-actinin, and actin cytoskeleton. Catulin may act as a cytoskeletal linker protein to modulate cell migration.

[0031] The inventors have discovered that catulin has a role in modulating tumor invasion and migration in vivo. As detailed in the Examples, catulin is highly expressed at the leading edge and in the invasive streams of cells in the malignant human HNSCC and in a mouse model of oral SCC. In vitro data show that an upregulation of catulin expression correlates with the transition of tumor cells from an epithelial to mesenchymal morphology and an increased expression of EMT markers vimentin and snail. Knockdown of catulin in hHNSCC cell lines dramatically decreases the migratory and invasive potential of those cells in vitro and metastatic potential in xenotransplants in vivo. Transcriptional and biochemical analyses of tumors deficient in catulin showed that its ablation prevented tumor cells from invading the surrounding stroma; this was accompanied by a decrease in expression of genes involved in cell migration and invasion such as integrins and Met receptor. These findings highlight the importance of catulin in SCC metastasis in vivo.

[0032] In an aspect, provided are methods of detecting metastatic cancer in a subject. The method may include measuring an amount of catulin in a sample from the subject, and comparing the amount of catulin in the sample to an amount of catulin in a control.

[0033] An increased level of catulin in the sample relative to the amount in the control may indicate metastatic cancer in the sample. The expression of the gene may be increased relative to the expression level of a control by an amount of at least about 1 -fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11 -fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40- fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, at least about 500-fold, or at least about 550-fold.

[0034] A subject can be an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orangutan, gibbon), or a human. In some embodiments, the subject is a mammal. In further embodiments, the mammal is a human.

[0035] As used herein, the term "sample" or "biological sample" relates to any material that is taken from its native or natural state, so as to facilitate any desirable manipulation or further processing and/or modification. A sample or a biological sample can comprise a cell, a tissue, a fluid (e.g., a biological fluid), a protein (e.g., antibody, enzyme, soluble protein, insoluble protein), a polynucleotide (e.g., RNA, DNA), a membrane preparation, and the like, that can optionally be further isolated and/or purified from its native or natural state. A "biological fluid" refers to any a fluid originating from a biological organism. Exemplary biological fluids include, but are not limited to, blood, serum, plasma, and colonic lavage. A biological fluid may be in its natural state or in a modified state by the addition of components such as reagents, or removal of one or more natural constituents (e.g., blood plasma). A sample can be from any tissue or fluid from an organism. In some embodiments, the sample comprises tissue from the digestive tract, lung, lip, mouth, esophagus, urinary bladder, prostate, lung, vagina, and/or cervix. In some embodiments the sample is from a tissue that is part of, or associated with, the skin of the organism. In some embodiments the sample comprises epidermis from the organism. In some embodiments the sample comprises epithelial cells from the organism. In some embodiemtns, the sample may be tissue from a neoplasm. A neoplasm may include cancer. In some embodiments, the sample may be cancerous tissue or from a tumor. In some embodiments, the sample may comprise tissue surrounding cancerous tissue or a tumor. In some embodiments, the sample may comprise tissue surrounding or around the perimeter of cancerous tissue or a tumor that was surgically excised. The cancer may comprise squamous cell carcinoma (SCC). The cancer may comprise melanoma. The cancer may comprise breast cancer. Malignant squamous cell neoplasms may include, for example, papillary carcinoma, verrucous squamous cell carcinoma, papillary squamous cell carcinoma, squamous cell carcinoma, large cell keratinizing squamous cell carcinoma, large cell nonkeratinizing squamous cell carcinoma, small cell keratinizing squamous cell carcinoma, spindle cell squamous cell carcinoma, adenoid/pseudoglandular squamous cell carcinoma, intraepidermal squamous cell carcinoma, lymphoepithelial carcinoma, basaloid squamous cell carcinoma, clear-cell squamous-cell carcinoma, keratoacanthoma, and signet-ring-cell squamous-cell carcinoma.

[0036] The amount of catulin in the sample may be measured by catulin antigen-antibody binding. In some embodiments, the gene expression of catulin may be evaluated. The methods described herein can include any suitable method for evaluating gene expression. Determining expression of at least one gene may include, for example, detection of an RNA transcript or portion thereof, and/or an expression product such as a protein or portion thereof. Expression of a gene may be detected using any suitable method known in the art, including but not limited to, detection and/or binding with antibodies, detection and/or binding with antibodies tethered to or associated with an imaging agent, real time RT-PCR, Northern analysis, magnetic particles (e.g., microparticles or nanoparticles), Western analysis, expression reporter plasmids, immunofluorescence, immunohistochemistry, detection based on an activity of an expression product of the gene such as an activity of a protein, any method or system involving flow cytometry, and any suitable array scanner technology. For example, the expression level of a protein may be evaluated by immunofluorescence by visualizing cells stained with a fluorescently-labeled protein-specific antibody, Western blot analysis of protein expression, and RT-PCR of protein transcripts. The antibody or fragment thereof may suitably recognize a particular intracellular protein, protein isoform, or protein configuration.

[0037] As used herein, an "imaging agent" or "reporter" is any compound or composition that enhances visualization or detection of a target. Any type of detectable imaging agent or reporter may be used in the methods disclosed herein for the detection of an expression product. Exemplary imaging agents and reporters may include, but are not limited to, compounds and compositions comprising magnetic beads, fluorophores, radionuclides, and nuclear stains (e.g., DAPI), and further comprising a targeting moiety for specifically targeting or binding to the target expression product. For example, an imaging agent may include a compound that comprises an unstable isotope (i.e., a radionuclide), such as an alpha- or beta- emitter, or a fluorescent moiety, such as Cy-5, Alexa 647, Alexa 555, Alexa 488, fluorescein, rhodamine, and the like. In some embodiments, suitable radioactive moieties may include labeled polynucleotides and/or polypeptides coupled to the targeting moiety. In some embodiments, the imaging agent may comprise a radionuclide such as, for example, a radionuclide that emits low-energy electrons (e.g., those that emit photons with energies as low as 20 keV). Such nuclides can irradiate the cell to which they are delivered without irradiating surrounding cells or tissues. Non-limiting examples of radionuclides that are can be delivered to cells may include, but are not limited to, ³⁷ Cs, ¹⁰³ Pd, ¹¹ ln, ²⁵ l, ²¹¹ At, ¹² Bi, and ²¹³ Bi, among others known in the art. Further imaging agents may include paramagnetic species for use in MRI imaging, echogenic entities for use in ultrasound imaging, fluorescent entities for use in fluorescence imaging (including quantum dots), and light-active entities for use in optical imaging. A suitable species for MRI imaging is a gadolinium complex of diethylenetriamine pentacetic acid (DTPA). For positron emission tomography (PET), ¹⁸ F or ¹¹ C may be delivered. Other non-limiting examples of reporter molecules are discussed throughout the disclosure. In some embodiments, determining the expression level of at least one gene includes measuring the expression level of an RNA transcript of the at least one gene, or an expression product thereof. In some embodiments, measuring the expression level of the RNA transcript of the at least one gene, or the expression product thereof, includes using at least one of a PCR-based method, a Northern blot method, a microarray method, and an immunohistochemical method.

[0038] The expression level of catulin in a sample may be compared to a control value associated with that same gene. A control may include comparison to the level of expression in a control cell, such as a non-cancerous cell or other normal cell. The control may be from a non-cancerous or normal cell from the same subject, or it may be from a different subject. Alternatively, a control may include an average range of the level of expression from a population of normal cells. Those skilled in the art will appreciate that a variety of controls may be used. In some embodiments, the control value associated with each gene may be determined by determining the expression level of that gene in one or more control samples, and calculating an average expression level of that gene in the one or more control samples, wherein each control sample is obtained from normal or healthy tissue of the same or a different subject.

[0039] Metastasis, a crucial step for tumor cells to invade distant tissues, has been the target for cancer therapy as it is responsible for over 90% of cancer-related deaths. Catulin is mapped in the human chromosome to a region where frequent mutations occur in a number of tumor types, including bladder, ovarian, esophageal SCC and testicular cancer and lymphomas. As shown herein, catulin knockdown dramatically decreases the invasive and metastatic potential of tumor cells. In some aspects, catulin may be used as a target for cancer therapeutics. In some embodiments, provided are methods of treating metastatic cancer. The method may include administering to a subject an effective amount of a therapeutic composition adapted to reduce the amount or the activity of catulin. The therapeutic composition may reduce or inhibit tumor metastasis. [0040] Inhibitors encompass agents that inhibit the activity of catulin. The amount or the activity of catulin may be reduced or inhibited using a variety of techniques known in the art. For example, an inhibitor may indirectly or directly bind and inhibit the activity of catulin, including binding activity or catalytic activity. An inhibitor may prevent expression of the catulin, or inhibit the ability of catulin to interact with cellular and extracellular components, moderate cell migration, and/or act in Rho signaling. For example, a therapeutic composition adapted to reduce the amount or the activity of catulin may comprise a small molecule inhibitor of catulin itself or of a binding partner, an antibody specific for catulin, or an siRNA. In some embodiments, the therapeutic composition may comprise an siRNA adapted to reduce the expression of catulin.

[0041] Compositions may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.

[0042] In a further aspect, provided are methods of determining a perimeter of a metastatic tumor. The method may include obtaining a sample of the tumor or surrounding tissue, measuring an amount of catulin in the sample, and comparing the amount of catulin in the sample to an amount of catulin in a control. An increased amount of catulin in the sample relative to the amount in the control may indicate the sample is part of the tumor. A substantially equivalent amount of catulin in the sample relative to the amount in the control may indicate the sample is part of non-tumor tissue beyond the perimeter of the tumor.

[0043] In a further aspect, provided are methods for monitoring progression of cancer in a subject undergoing therapeutic treatment. The method may include measuring an amount of catulin in a first and a second sample taken from the subject at a first and a second time, and comparing the first and second amounts. An increase in the amount of catulin in the second sample relative to the amount in the first sample may indicate a progression of the cancer. [0044] In a further aspect, provided are methods for determining the aggressiveness of post-operative cancer therapy for a subject. The method may include obtaining a sample of a tumor from the subject, measuring an amount of catulin in the sample, and treating the subject post-operatively. The aggressiveness of post-operative treatment may be dependent on the amount of catulin measured in the sample.

[0045] Modeling tumor cell invasion the way it occurs in the body, i.e., monitoring three dimensional movements of cells through ECM (Extracellular Matrix), can be difficult and conventional in vitro studies of this process might be misleading. One major difficulty is caused by the transient, reversible nature of EMT during carcinoma invasion and metastasis, and so it is challenging to observe EMT in human carcinomas. As detailed herein, however, we discovered a new biomarker, a-catulin, whose expression and function correlates with invasive behavior of carcinoma.

[0046] Using a human catulin promoter fragment driving GFP expression in a HNSCC and breast cancer xenotransplant system, we developed a reporter system which for the first time allows isolation and characterization of invasive cells at the tumor front. Using this system a proportion of carcinoma cells at the invasive tumor front can be isolated and visualized for functional, genetic, and epigenetic characterization. Unlike conventional transcriptional profiling, which does not allow for characterization of a pure population of cells isolated directly from an in vivo model, and wherein gene expression alterations in invasive cells are often masked by the majority of non-metastatic cells, the system described herein allows detection of such transient cells in vivo in progressing tumors. The system may also be used as a tool to assess the contribution of EMT to the behavior of high grade carcinomas. There is a strong need to characterize cells in vivo during tumor progression that begin to go through partial or complete EMT and invade surrounding stroma. Using the system described herein could lead to development of early detection markers of invasion and more importantly develop targeting strategies against invasive tumor cells.

[0047] In some aspects, provided is a reporter system. The reporter system may comprise a recombinant cell. In some embodiments, provided is a recombinant cell whose genome comprises a first polynucleotide corresponding to the 5'-flanking sequence located from about 1 .3 kb upstream and to about 200 bp downstream of the human a-catulin gene transcription start site, the first polynucleotide upstream up and driving the expression of a second polynucleotide encoding a fluorescent polypeptide. The fluorescent polypeptide may comprise any fluorescent polypeptide known in the art, such as, for example, GFP, RFP, and YFP. The cell may be a human SCC cell line or breast cancer cell lines. The SCC cell line may comprise, for example, SCC1 5, SCC25, or USC-HN 1 cell lines, and breast cancer cell lines may comprise, for example, MDA-MB-231 . The cells may be grown and proliferated. The cells may be separated and isolated using FACS. The cells may be functionally tested and characterized genetically and epigenetically and compared to noninvasive cancer cells. The recombinant cells may be injected into mice.

[0048] Further described herein is a three dimensional tumor spheroid-based functional assay for target validation. The three dimensional assay may include a recombinant cell as described above. This functional test combined with data obtained using primary human cancer specimens may be used to develop potential new markers of invasion and novel targets for anti- metastasis therapeutics. In some embodiments, the recombinant cell may be grown in a composition comprising a medium such as, for example, collagen, matrigel, fibronectin, and laminin. The composition may be spherical in shape. Growth and/or migration of the recombinant cells beyond the spheres may be observed and analyzed after treatment with a small molecule library or shRNA library. In some embodiments, provided are methods of testing a compound for anti-metastasis activity. The method may include growing a recombinant cell whose genome comprises a first polynucleotide corresponding to the 5'-flanking sequence located from about 1 .3 kb upstream and to about 200 bp downstream of the human a-catulin gene transcription start site, the first polynucleotide upstream up and driving the expression of a second polynucleotide encoding a fluorescent polypeptide. The recombinant cell may be grown in a medium. The method may further include contacting the recombinant cell with the compound, measuring at least one of growth and invasion of the recombinant cell into the medium, comparing the at least one of growth and invasion with a control, and determining the antimetastasis activity of the compound. A reduction in the at least one of growth and invasion relative to the control may indicate antimetastasis activity of the compound. A control may include comparison to the level of growth and/or invasion of the recombinant cell in the medium without being in the presence of the compound. Alternatively, a control may include an average range of the level of growth and/or invasion from a population of recombinant cells. Those skilled in the art will appreciate that a variety of controls may be used.

[0049] As a-catulin expression and function correlates with early onset of tumor cell invasion, the reporter system developed using the catulin promoter in the human SCC and breast cancer xenotransplant model will facilitate the marking, tracking, and isolation of a small minority of carcinoma cells that may be invasive and undergo an EMT in primary tumors. Further, the model may facilitate the characterization of new diagnostic markers of invasion and also to understand early signaling pathways involved in tumor invasion for future therapy development.

[0C50] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims.

EXAMPLES

Example 1 : Materials and Methods.

[0051] Generation of stable cell lines and cell culture, a-catenin keratinocyte cells were cultured as previously described. SCC-15 and USC-HN1 cells were cultured in DMEM High Glucose (Gibco) supplemented with 10% FBS, 1001 U penicillin and 1001 -pg/mL streptomycin. GIPZ and TRIPZ lentiviral shRNA clones are available from Open Biosystems and packaged according to manufacturer's protocol. At 48 hours post-transduction, cells were selected with puromycin to establish stable cell lines.

[0052] Xenograft transplants. 1 x10 ⁶ cells in media were mixed 1 :1 with matrigel (BD Biosciences) and injected subcutaneously between the neck and shoulder of NOD.Cg mice. Tumors were allowed to form for 4-9 weeks before sacrificing and collecting the primary tumor, metastatic cells and lungs from each subject. Tumors and metastatic cells used for RNA isolation were first dissected from the mouse, minced into small pieces, incubated in collagenase (1000 U/mL) for 1 hour at 37°C, washed in PBS, trypsinized (0.25% trypsin-EDTA from Gibco) for 20 mins at 37°C, neutralized and FACS sorted for GFP+ cells using a BD Biosciences FACSAria cell sorter. [0053] RNA Isolation, Microarray, RT-PCR. a-catenin WT and KO keratinocytes in culture were collected in TRizol® reagent (Invitrogen) and total RNA extracted using the RNeasy® Mini Kit (QIAGEN). FACS sorted cells were immediately collected in RNAprotect® cell reagent (QIAGEN) before spinning down and resuspending in TRizol® (Invitrogen). Microarray analysis was performed by the University of Southern California Microarray Core Facility using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays. To perform semi-quantitative RT-PCR, RNA was reverse-transcribed to cDNA with Superscript™ II (Invitrogen). Primer sequences are given in SI Methods.

[0054] Western Blot. Cells were washed and scraped in cold PBS. Cell pellet from a 10cm plate was resuspended in 2001 -pL of RIPA lysis buffer containing 0.2% SDS, 10 mM NaF, 1 mM Na ₃ V0 ₄ and protease inhibitor cocktail III (Ca!biochem). Cells were passed through a 21 g needle, gently rotated for 30 mins at 4°C and spun down at 14,000xg for 15 min at 4°C. Protein lysates were collected and separated on 4%-12% NuPAGE Novex Bis-Tris gels (Invitrogen) and blotted onto a nitrocellulose membrane with a semi-dry transfer system (Hoeffer TE77XP) for 1.5 hours at 75 mA. Membrane was blocked in 5% skim milk in TBS containing 0.1 % Tween-20 (TBS-T) for 30 mins at RT on a gentle rocker. Membrane was than incubated with primary antibody in 5% skim milk in 0.1 % TBS-T with gentle rocking overnight at 4°C. Antibodies are described in SI Methods. LI-COR IRDye® infrared-conjugated secondary antibodies were diluted 1 :10,000 in 5% skim milk in 0.1 % TBS-T. Signals were detected by the Odyssey Infrared Imaging System (LI-COR Biosciences ).

[0055] Indirect Immunofluorescence Detection. Tumors from our xenograft animal models were dissected and immediately embedded in OCT and sectioned at 10 μΜ for indirect detection of various markers. Samples were fixed in 4% PFA for 10 mins and subsequently permeabilized in 0.1 % Triton X-100 in PBS (PBS-T) for 10 mins. Next, samples were blocked in 0.1 % BSA, 2.5% HI-GS, 2.5% HI-DS in 0.1 % PBS-T for 30 mins at RT. Primary antibodies were diluted in 0.1 % BSA in 0.1 % PBS-T and incubated overnight at 4°C. Alexa Fluor 488 or 594 secondary antibodies were diluted 1 :500 in blocking solution and incubated 1 hour at RT. Photographs were taken using Axiolmager Z1 (Zeiss). Primary antibody descriptions and dilutions are described in SI Methods.

[0056] Immunohistochemistry. Tumors from our xenograft animal models were fixed in 4% PFA overnight at 4°C, washed well in PBS, ethanol dehydrated, embedded in paraffin and sectioned 6 μΜ thick. Human head and neck SCC tissue array is available from US Biomax. Samples were deparaffinized and pretreated using antigen retrieval 2100 Retriever (Proteogenix). Endogenous peroxidase was blocked in 0.03% hydrogen peroxide for 5 mins, washed in 0.3% PBS-T, blocked in 0.1 % gelatin, 2.5% HI-GS, 2.5% HI-DS, 0.1 % BSA in 0.3% PBS-T for 1 hour and incubated with primary antibody in 0.1 % BSA in 0.1% PBS-T overnight at 4°C. After washing well in 0.3% PBS-T, biotinconjugated secondary antibodies (Vector Laboratories) were diluted 1 :100 in blocking solution and incubated 1 hour at RT. The ABC Kit was then used following manufacturer's instructions (Vector Laboratories). Staining was detected using DAB Peroxidase Substrate Kit following manufacturer's instructions (Vector Laboratories). Antibodies are described in SI Methods.

[0057] β-galactosidase Detection. To detect β-galactosidase activity, sections were fixed in 0.2% glutaraldehyde for 2 mins, washed well with PBS and stained overnight at 37°C with X- Gal staining solution (5 mM EGTA, pH 8, 2 mM MgCI ₂ , 0.2% NP-40, 0.1% sodium deoxycholate, 2 mM CaCI ₂ , 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg/mL X-Gal in PBS).

Example 2: Catulin is upregulated in metastatic cells.

[0058] It was previously shown that the conditional loss of cell-cell junction protein a-catenin in skin results in the formation of SCC-like phenotype, accompanied by increased cell proliferation and motility. Microarray expression profiling of the a-catenin cKO cells showed an upregulation of the α-catenin homologue a-catenin-like 1 , catulin. We verified the upregulation of catulin in the motile α-catenin KO cells by RT-PCR using RNA from pure fractions of acatenin WT and KO keratinocytes (Fig. 1A).

[0059] As the loss of a-catenin from cell-cell junctions in human tumors correlates with EMT and increased invasiveness, we wanted to test if the in vitro data correlates with in vivo tumor metastasis. We generated a xenograft metastasis model where USC-HN1 , highly metastatic hSCC cells, were labeled with GFP for tracking and injected to NOD.Cg mice. After 5 weeks, as visualized by GFP, the injected hSCC cells formed primary tumors and had spread and metastasized to distant tissues including lymph nodes and lungs, predominantly through the lymphatic vessels (Fig. 1B). Carefully dissecting out the primary tumor and metastatic cells, we FACS sorted for and collected GFP+ cells from the two populations (Fig. 1C). RNA was isolated from these GFP+ cells and used for microarray expression profile analysis. One of the upregulated genes in the metastatic cells compared to the primary tumor was catulin. This upregulation was confirmed on independent samples by RT-PCR (Fig. 1 D).

[0060] To verify that the upregulation of catulin correlates with EMT in human SCC, we induced those cells that display an epithelial morphology to undergo EMT with various growth factors, TGF-β, EGF and Wnt3a, alone and in combination (Fig. 1 E). Observing the cellular morphology, EMT induction was successful in these cells. Upon looking at the expression profiles of these cells, we saw upregulated catulin expression in the cells that had undergone EMT (Fig. 1 F). Increased catulin expression in the cells correlated with known mesenchymal markers, vimentin and snail. The in vitro data agrees with the in vivo data wherein catulin is upregulated in more motile, mesenchymal cells.

EXAMPLE 3: Catulin is expressed at the leading edge and in invasive, metastatic streams of cells in SCC.

[0061] Upon seeing catulin upregulated in the mouse model of metastasis in vivo and in EMT-induced mesenchymal cells in vitro, we wanted to see how this correlated with human SCCs. In a tissue array consisting of normal oral mucosa and various grade human HNSCCs, catulin showed low levels of expression in the normal mucosa epithelium, and although catulin expression varied slightly in level and pattern in the malignant grade sees, in 19 out of 22 cases, it mostly localized to the leading edge of the primary tumor (Fig. 2A-F). In higher grade SCCs that had streams of cells metastasizing away from the primary tumor, catulin was strongly expressed in those motile, invasive cells (Fig. 2D'-F').

[0062] To be certain that the antibody staining results truly reflect catulin expression, we employed a mouse model of 4NQO-induced tongue SCC on transgenic catulin-P-galactosidase reporter mouse line generated in our laboratory, where the β-galactosidase reporter gene is under the control of endogenous catulin promoter. Mice were administered 4-nitroquinoline- oxide (4NQO), a DNA adduct-forming compound that serves as a surrogate to tobacco exposure, to induce oral SCC. When visualizing catulin expression, by x-gal staining, in the 4NQO-induced SCC after 22 weeks, it is strongly expressed at the leading edge of the epithelium where it invaginates into the dermis (Fig. 2H). 28 weeks after initial 4NQO administration, we observe deeper invagination of the epidermis, correlating with the carcinogenic progression, and increased catulin expression (Fig. 21). Catulin expression is increased in 4NQO-induced SCCs compared to low basal levels of expression in non-treated controls (Fig. 2G). This data in our animal model correlates with the human tumor tissue array that was analyzed, where catulin has increased expression in sees and additionally, is expressed in the leading edge of the tumor.

EXAMPLE 4: Catulin-deficient tumor cells are unable to efficiently migrate and invade in vitro.

[0063] Upon seeing the upregulation of catulin in more mesenchymal, metastatic SCC cells, we wanted to test the role of this protein in tumor cell migration and invasion. To obtain stable hSCC cell lines deficient of catulin, we generated lentivirus containing catulin-specific shRNA to knockdown catulin. The construct also contained a turboGFP reporter for visualization of cells transduced with the shRNA (Fig. 3A). We were able to successfully knockdown catulin in hSCC cell lines (G85) compared to cells transduced with the non-silencing control (GNS) on the RNA and protein level (Fig. 3B-C). We confirmed the specific knockdown in two hSCC cell lines, SCC-15 and USC-HN1.

[0064] In vitro migration and invasion assays using those cell lines showed that catulin ablation in SCC cells decreased the ability of these cells to migrate and invade respectively (Fig. 3D and Fig. 3E). Analyzing these cells for potential differences in proliferation rate, we performed a growth curve and did not see a difference between control and catulin-deficient cancer cells (Fig. 8A).

[0065] To address previous findings that catulin-deficient cells are more apoptotic, we analyzed control and catulin-deficient SCC cells for AnnexinV activity under normal culture conditions and after induction of apoptosis (Fig. 8B). We do not see a significant difference in AnnexinV profiles between the two cell populations. It is possible that the discrepancy between what we see here and what was previously reported may be due to different cell lines used or differences in levels of catulin knockdown.

EXAMPLE 5: Ablation of catulin in hSCC cell lines decrease their metastatic potential in vivo.

[0066] Catulin-ablated hSCC cells are less migratory and invasive in vitro; we wanted to test if our observations are relevant in vivo. Non-silencing control and catulin knocked down hSCC cells were injected subcutaneously in the neck area into NOD.Cg mice and the tumors that formed after 5-9 weeks were collected for analysis. Both SCC-15 and USCHN1 cell lines were initially tested, but since SCC-15 cell line showed poor metastatic potential, it was excluded from further in vivo metastasis assays. Overall, we observed that tumors from the control GNS and catulin knockdown G85 cells were similar in size (Fig. 4A). We can attribute this to similar growth rates of the two cell lines (Fig. 4B). Our observations were consistent when using a separate system with an independent catulin-specific shRNA, in which catulin knockdown occurred only after doxycycline induction (Fig. 7A-E).

[0067] Upon injecting these cells into mice, we can visualize tumor formation and track metastasis with the turboGFP reporters in vivo. We found that subcutaneous injection of the tGFP labeled tumor cells in the neck area is a very useful assay that allows us to quantitatively assess in vivo metastatic potential of tumor cells by counting new foci in the lungs. We observed that catulin-ablated tumor cells are dramatically less metastatic (Fig. 4C-L). When this is depicted graphically (Fig. 4M), on average, catulin-deficient SCC tumor cells are half as metastatic as control SCC tumor cells (n=6). This data correlates well with our in vitro data where the ablation of catulin in tumor cells results in a decrease in the migratory and invasive potential of the cells. This data was mirrored in our inducible system where catulin-ablated cells are less metastatic than control cells, about three-fold in these cell lines (n=6) (Fig. 6F-N).

EXAMPLE 6: Signaling pathways changed by the loss of catulin.

[0068] To better understand how the ablation of catulin in tumors lead to decreased capability to metastasize, we dissected the control GNS and catulin-deficient G85 tumors, FACS sorted for GFP+ pure population of epithelial tumor cells, and isolated RNA from them for microarray expression profiling. We performed functional annotation of the microarray data using Ingenuity Pathways Software to identify the biological functions that were significantly represented in the catulin-deficient tumors. Cellular movement, including invasion and migration, was amongst the 10 most enriched categories (Table 1).

Table 1. Genes involved in cellular movement differentially expressed in catulin-deficient tumor. Genes in bold-underline are upregulated whereas the other genes are downregulated in a catulin-deficient tumor compared to control tumor.

Movement DPP4, ENPP2, ETS1 , ETV1 , FAM5C, HIF1A, HIPK2,

ID2. IGF1 R, IQGAP1 , ITGA2, ITGA6, ITGB1 , KITLG, LGR4. MARCKS. MET. MMP1. FE2L2. PDCD4, PICALM, PRKAA1 , PTGS2, PTK2, PTP4A1 , SEL1 L, SERPINE1. SP100. TNFSF10. XIAP

Cellular Migration 6.43E-04 ADAM 10. AGK. ASAP1. BCAR1. C50RF13. CASP8. Movement CD44. CDKN1 B. LIC4. COL18A1. CTBP2. CTTN.

DBF4. DEFB1. DPP4. DPYSL2. ENPP2, ETS1 , FAM5C, FOX03, GNA13, HIF1A, IGF1 R, IQGAP1 , ITGA2, ITGA6, ITGB1 , KITLG, MAP3K7, MET, MLL, PTGS2, PTK2, PTP4A1 , PTPN11 , RANBP9, REPS2, RLN2. SAA1. SERPINE1. SP100. TGM2. TMOD3. TNFAIP8, TNFSF10, LAMB1

[0069] Catulin-deficient tumors that are unable to metastasize showed a decrease in integrin expression, specifically ITGA2, ITGA6, and ITGB1, and additionally, a decrease in genes involved in integrin signaling like: ASAP1, BCAR1, PTK2, PIK3CA, PARVA, ACTR2, CTTN, and LAMB1.

[0070] Interestingly, many of the genes decreased in catulin-deficient tumors are involved in Met/HGF signaling pathway. Those genes include: MET, ELF1, ETS1, KRAS, MAP3K7, PIK3CA, PTGS2, PTK2, and PTPN11. HGF/Met signaling was shown to contribute to oncogenesis and tumor progression in several human cancers and promote aggressive cellular invasiveness that is strongly linked to tumor metastasis.

EXAMPLE 7: Catulin-deficient hSCC tumor cells are unable to rearrange the ECM for invasion in vivo.

[0071] To verify the changes observed in the microarray and further analyze catulin- deficient tumors that were unable to metastasize to distant tissues, we dissected out the tumors that formed from control GNS- and catulin-deficient G85-injected cells and analyzed them by immunofluorescence. Studying the tumor morphology, we noticed that the control tumor has streams of invasive cells, whereas catulin-deficient tumors do not (Fig. 5A-B). Initially, we wanted to confirm localization of catulin in the normal tumor and its absence in the catulin- deficient tumors by staining with catulin antibody (Fig. 5C-D). In the control tumor, catulin was expressed at the migratory front and in the invasive streams of cancer cells, similar to what was seen in the human HNSCC tissue array (Fig. 2B-F). Catulin was indeed absent in the tumors that formed from injection of catulin-deficient cells. Samples were also stained with the proliferation marker Ki67 to exclude differences in tumor growth; similar cell proliferation was observed (Fig. 8A-B). This correlates with the growth curves of these cells (Fig. 4B).

[0072] Catulin-deficient tumors lacked streams of invasive cells when compared to control tumors; we therefore analyzed the tumors for EMT with vimentin, a mesenchymal marker (Fig. 5E-F). The control tumor has small groups of cells that express vimentin, whereas in catulin- ablated tumors, there was no vimentin expression (Fig. 5E-F).

[0073] We further analyzed the tumors that formed from the injection of control and a-catulin deficient USC-HN1 cell lines. We observed and tracked injected cells by the tGFP reporter. As shown in Figure 16A and Figure 16B, checking for proliferation rate, we did not see a considerable difference between the two tumors, as assessed by the proliferative marker, Ki67. As shown in Figure 16C and Figure 16D, when analyzing the tumors for human-specific lymphatic vessel marker, lyve-1 , the control tumor (Figure 16C) had collective groups of cells that were able to invade the surrounding stroma and form their own lymphatic vessels, whereas a-catulin-deficient tumors (Figure 16D) were unable to do so. IHC staining using phospho-JNK antibody show an increase in activated phospho-JNK in control tumor (Figure 16E) compared to a-catulin deficient tumor (Figure 16F).

[0074] As our array data showed that integrin signaling was affected and it is known that integrins are involved in bidirectional signaling, resulting in ECM rearrangement and transduction of ECM signals to achieve cell movement, we focused our analysis on integrin and ECM components in catulin-deficient tumors. Examining (34 integrin, a marker of the basal layer, we see small streams of cells in the control tumor that express β4, but in the catulin- deficient tumor, it is only expressed in the solid tumor (Fig. 5G-H). Looking at laminin, a component of the ECM, the invasive front of the control tumor expresses laminin where it is aligned with the streams of invasive tumor cells, whereas in the catulin-ablated tumor, cells express laminin minimally and it is still surrounding the solid mass of tumor (Fig. 5I-J). [0075] We also noticed that control tumors express more lyve-1 , a marker of lymphatic vessels than catulin knockdown tumors, indicating that the control tumor is more lymphogenetic, able to invade the surrounding stroma and create their own lymphatic vasculature network (Fig. 7E-F).

[0076] Because we observed a decrease in Met/HGF signaling pathway genes that can transduce signals to MAP kinase, which have been implicated in cell migration in a number of studies, we looked at the MAPK family member J. When examining the tumors that formed, we observed an increase in activated phosphorylated JNK in the control tumors at the leading edge (Fig. 5K-L). This suggests that the downregulation of phospho-JNK may indirectly play a role in decreased cell migration and subsequent metastasis observed in catulin-deficient tumors.

EXAMPLE 8: a-catulin-GFP reporter system reflects the expression of endogenous a- catulin in the invasive front of SCC and might be useful to isolate this small population of cells from the tumor.

[0077] The putative human a-catulin gene promoter was partially characterized. To generate a catulin reporter system, a promoter clone containing a 1.5 kb insert corresponding to the 5' -flanking sequence located approximately 1.3 kb upstream and up to 200 bp downstream of the human a-catulin gene transcription start site (TSS) was placed upstream of GFP. We generated a xenograft model where 3 different hSCC (SCC15, SCC25, and USC-HN1) cell lines stably expressing catulin-GFP reporter, were injected to immuno-compromised NOD.Cg mice. After 5 weeks, the injected hSCC cells formed primary SCC tumors (Fig. 9). The GFP positive cells were observed only in some areas of the tumors - possibly invasive foci (Fig. 9B) and it was consistent in tumors formed from injection of all 3 cell lines.

[0078] To confirm that the expression of GFP correlated with the endogenous expression of catulin, we stained the slides with α-catulin antibody. Indeed the GFP positive stream of invasive cells overlapped with the staining for catulin (Fig. 10) which confirms the correct function of the reporter system.

[0079] To verify the localization of catulin-GFP positive cells within the tumor we dissected out and analyzed the tumors that formed after injection of SCC cell lines stably expressing the catulin-GFP reporter (Fig. 11). GFP positive cells were localized at the invasion front and in the streams of cancer cells (Fig. 11B and Fig. 11E), similar to what was observed previously in the human SCC tissue array stained with catulin Ab (Fig. 12). Arrows indicate GFP positive cells (Fig. 11B and Fig. 11E) in the tumor where integrin beta4 staining is no longer sharply separating tumor from stroma (Fig. 11C and Fig. 11F) indicating the invasive character of those cells.

[0080] As detailed above, we already tested that we are able to sort those cells out of the tumor for further analyses. Carefully dissecting the areas of tumor that were enriched for the GFP positive cells (Fig. 9B) we sorted by FACS for and collected GFP+ cells using integrin alpha-6 as an additional epithelial marker for purity (Fig. 13).

EXAMPLE 9: The invasive behavior of cells isolated using the catulin reporter system.

[0081] We will utilize a xenograft model in which 3 different hSCC (SCC15, SCC25 and USC-HN1) cell lines stably expressing Catulin-GFP reporter will be injected to immunocompromised NOD.Cg mice as previously described (Fig. 9). To isolate the Catulin-GFP positive cells we will use a combination of enzymatic digestion and flow cytometry. The tumors will be subjected to enzymatic digestion with collagenase I (1000 U/mL) for 1 hour at 37°C followed by 0.25% trypsin digestion for 15 min at 37°C. Single cell suspensions will be analyzed or sorted directly. We will use GFP and integrin alpha-6 as an additional epithelial marker for purity. Isolated GFP+/alpha6+ (invasive cells) and alpha6+ (remaining non-invasive tumor cells - control) from tumors formed after injection of 3 different hSCC (SCC15, SCC25 and USC-HN1) cell lines stably expressing Catulin-GFP reporter will be used for additional experiments.

[0082] Expression of known EMT markers. FACS sorted cells [GFP+/alpha6+ (invasive cells) and alpha6+ (control)] will be immediately collected in RNAprotect® cell reagent (QIAGEN) before spinning down and resuspending in TRIzol® (Invitrogen). Total RNA from FACS sorted cells will be extracted using RNeasy® Micro Kit (QIAGEN). To perform semiquantitative RT-PCR and quantitative real-time PCR, RNA will be reverse-transcribed to cDNA with Superscript™ II (Invitrogen). Real- time qPCR will be performed on an ABI 7900HT Fast Real-Time PCR System using primers for known EMT markers: SNAIL, SLUG, TWIST, ZEB1 , ZEB2, VIMENTIN, NCADHERIN, and ECADHERIN.

[0083] Assessments of the morphology of catulin-GFP+ cells. After isolating and sorting the [GFP+/alpha6+ (invasive cells) and alpha6+ (control)] cells we plan to plate them in culture to observe their cellular morphology and perform immunofluorescent staining using epithelial and mesenchymal markers like Ecadherin, Beta-catenin, Vimentin to confirm their mesenchymal morphology.

[0084] Migration and invasion potential of catulin GFP+ cells. FACS sorted cells [GFP+/alpha6+ (invasive cells) and alpha6+ (control)] will be used for in vitro migration and invasion assays. BD Falcon cell culture inserts will be used for migration assays and BD BioGoat™ Matrigel™ invasion chambers will be used for invasion assays. 200,000 cells will be counted and resuspended in serum-free media and added to each chamber insert for both the migration and invasion assays. Media containing serum, providing a chemoattractant for the cells, will be added to the lower chamber. Cells will be cultured in a humidified tissue culture incubator and allowed to migrate and invade for 36 hours and 48 hours, respectively. Migration and invasion assays will be performed in duplicate and repeated independently 3 times. After the desired time point is reached, cells will be fixed in 5% glutaraldehyde, washed and stained with 0.5% Toluidine Blue. The number of cells that went through the porous membrane will be counted under a microscope using a 20x objective; cells will be counted for 5 fields for each membrane.

[0085] In vivo metastatic potential of catulin GFP+ cells. To test if catulin-GFP+ cells have higher metastatic potential in vivo we will perform tail vein injection assays. Isolated by FACS cells will be resuspended in phosphate-buffered saline (PBS) to a concentration of 5x10 ⁴ cells/mL. A total volume of 200 μΙ_ of cell suspension will be injected into the tail veins of 6 NOD.Cg mice per each group of cells [GFP+/alpha6+ (invasive cells) and alpha6+ (control)] per each cell line (SCC15, SCC25 and USC-HN1 ). After the injections, mice will be sacrificed at 3 months to evaluate for the presence of metastases. Lungs will be harvested, embedded in OCT, sectioned, and stained with hematoxylin and eosin (H&E) to confirm the presence or absence of metastases.

[0086] The cells isolated using the catulin-GFP reporter system will have higher invasion and migration potential both in vitro and in vivo as compared to GFP negative tumor cells. Those catulin-GFP+ cells will reflect the epithelial-mesenchymal transition phenotype as judged by the expression of known EMT markers and down regulation or cytoplasmic localization of cell-cell adhesion proteins like E-cadherin and Beta-catenin.

EXAMPLE 10: Analysis of invasive cells isolated using the catulin reporter and confirmation of greater tumor initiating potential and drug resistance.

[0087] We will test if invasive cells isolated using the catulin reporter have higher tumor initiating potential and drug resistance. Isolated GFP+/alpha6+ (invasive cells) and alpha6+ (remaining non-invasive tumor cells - control) from tumors formed after injection of 3 different hSCC (SCC15, SCC25 and USC-HN1) cell lines stably expressing Catulin-GFP reporter will be used for experiments.

[0088] Colony Formation Assay. To investigate the colony formation ability of sorted by FACS cancer cells [GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive control)] 1 ,000 cells will be seeded into a 10 cm plate. The cells will be then cultured in standard conditions and after one week, the plates will be washed, fixed with a 100% methanol and stained with Giemsa stain. The images will be taken and number of colonies will be counted.

[0089] Sphere Formation Assay. To test the sphere formation ability of sorted by FACS cancer cells [GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive control)] 100 cells per a well in a 6 well low attachment plate will be grown in DMEM Basal Medium with B-27 serum free supplement, 20 ng/mL basic human fibroblast growth 17 factor bFGF, 20 ng/mL human recombinant epidermal growth factor EGF and 4 μg/mL insulin. After one week, the images will be taken and number of spheres will be counted.

[0090] Drug Resistance. Sorted by FACS cancer cells [GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive control)] will be collected and re-plated at a density of 3,000 cells per a well in a 96-well plate. The next day, cisplatin and/or 5 FU will be added to the cells at a concentration of 0 μΜ, 1.5 μΜ, 3 μΜ, 4.5 μΜ, and 6 μΜ. The wells will be then analyzed by MTS assay (cell proliferation assay) 48 hours and 72 hours after treatment. Absorbance will be then read at 490 nm using the Hidex Multilabel Detection Program and MikroWin 2000 to analyze the data.

[0091] In vivo tumor initiation assay. Sorted by FACS GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive control) will be mixed with matrigel (1 :1 vol) and injected to Nod.Cg mice. We plan to use different amounts of cells per injection: 1000, 10 000, 50 000, 100 000 to compare tumorigenic potential of GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive control) cells.

[0092] In vivo drug resistance assay. To test if GFP+/alpha6+ (invasive cells) are more drug resistant than the alpha6+ (non-invasive control) cells in vivo we are planning to inject mice with 3 different hSCC (SCC15, SCC25 and USC-HN1) cell lines stably expressing the Catulin-GFP reporter. After 4 weeks when the tumors are already formed, mice will be treated with the chemotherapy agent 5 fluorouracil (5FU). In experiments, 50 mg/kg 5FU will be injected i.p. weekly for a total of three doses. Mice will be euthanized initially at week 1 , 2, 3, and 4 to assess the behavior of GFP+/alpha6+ (invasive cells) exposed to 5FU treatment. The number of cells will be analyzed using a LSR Flow cytometer analyzer. Samples will be also embedded in OCT and cryosections will be analyzed using proliferation (Ki67) and apoptosis (Active Caspase-3) markers.

[0093] Expected outcomes. GFP+/alpha6+ (invasive cells) will have higher colony and sphere formation potential as compared to non-invasive cells, be drug resistant both in vitro and in vivo, and have higher tumor initiating potential in vivo. These results would demonstrate that the EMT phenotype correlates with the tumor initiating potential of cells designated to form distant metastasis. Initiating cancer cells, so called "cancer stem cells" that have undergone EMT exhibit therapeutic resistance and may therefore also form a reservoir of surviving cells that is responsible for tumor recurrence after apparently successful initial therapy.

EXAMPLE 11 : Isolation and characterization of diagnostic markers of advance malignancy and signaling pathways involved in tumor invasion using the catulin reporter system.

[0094] Using human catulin promoter fragment driving GFP expression in our hSCC xenotransplant system, we will visualize and isolate a small minority of carcinoma cells at the invasive tumor front for genetic characterization.

[0095] Isolation of catulin-GFP+ invasive cancer cells for analysis. We will utilize a xenograft model in which 3 different hSCC (SCC15, SCC25, and USC-HN1 ) cell lines stably expressing Catulin-GFP reporter will be injected to immuno-compromised NOD.Cg mice as described above. To isolate the Catulin-GFP positive cells, we will use a combination of enzymatic digestion and flow cytometry. The tumors will be subjected to enzymatic digestion with collagenase I (1000 U/mL) for 1 hour at 37°C followed by 0.25% trypsin digestion for 15 min at 37°C. Single cell suspensions will be analyzed or sorted directly. We will use GFP and integrin alpha-6 as an additional epithelial marker for purity. FACS sorted cells [GFP+/alpha6+ (invasive cells) and alpha6+ (control)] will be immediately collected in RNAprotect® cell reagent (QIAGEN) before spinning down and resuspending in TRIzol® (Invitrogen). Total RNA from FACS sorted cells will be extracted using RNeasy® Micro Kit (QIAGEN). The GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive - control) cells samples will then be used to probe a human genome array U-133 2plus (Affymetrix) and then read at the CHLA Genomics Core facility and raw data will be analyzed with Partek Genomic Suite followed by Ingenuity Pathways Analysis software. Genes will be annotated into putative functional categories.

[0096] We will obtain a more accurate picture of signaling pathways active in invasive cancer cells and do comprehensive transcriptome and epigenome comparisons of GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive - control) to identify pathways and gene targets that are involved in cell invasion. Whole genome DNA methylation analysis will be completed. ChlP-seq experiments to study the differential modifications between GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive - control) cells on histone marks (H3K4me, H3K9Ac, H3K27Ac) will be completed. The whole transcriptome will be compared using RNA-seq for GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive - control) cells, and correlate the RNA-seq results with DNA methylation and ChlP-seq results.

[0097] Validation of array results by Real-time PCR. FACS sorted cells [GFP+/alpha6+ (invasive cells) and alpha6+ (control)] will be immediately collected in RNAprotect® cell reagent (QIAGEN) before spinning down and resuspending in TRIzol® (Invitrogen). Total RNA from FACS sorted cells will be extracted using RNeasy® Micro Kit (QIAGEN). To perform real-time PCR, RNA will be reverse-transcribed to cDNA with Superscript™ II (Invitrogen). Real-time qPCR will be performed on an ABI 7900HT Fast Real-Time PCR System using primers for selected genes obtained in our screens. Attention will be paid to those messages uniquely up- or down-regulated that are consistent between 3 different SCC lines as those may be the most universal markers of invasion. Those will become subject to validation by Real-time-PCR.

[0098] By comparing pure population of GFP+/alpha6+ (invasive cells) and alpha6+ (non-invasive control) we will define the genetic and epigenetic changes conferring invasive behavior of those cells. The identification of the downstream effector pathways that are activated during invasion could reveal new diagnostic markers of advanced malignancy and novel targets for anti-metastasis therapeutics.

EXAMPLE 12: Verification of array results using immunohistochemistry.

[0099] The genes that will be enriched in GFP+/alpha6+ (invasive cells) consistently in different SCC lines will be analyzed and validated by Real-time-PCR. To validate the data and check the correlation with human SCC we are planning to perform immunohistochemistry with antibodies against selected genes using primary tumor specimens obtained from the clinic. Collected specimens will consist of human SCC cancer specimens with adjacent normal tissue margin that have to be removed during scheduled surgeries. Tissue specimens only wiil be used to verify which of the newly characterized using reporter model markers are also present and where exactly they localize within the samples of malignant squamous cell carcinoma. On the day of scheduled surgery, tissues from the primary tumor and adjacent normal tissue will be collected. During surgery, immediately after resection of tumor, tissues will be sent for pathology review. After the pathology review is done the primary tumor tissue and adjacent normal tissue will be frozen in OCT in the operating room. The tissue samples will then be transferred to the research lab on dry ice. Specimens will be obtained in an "identity- blinded" manner, with the investigators in this study having access only to the transported tissue samples - patient identity will be de-linked from the database. Specimens will be sectioned at 10 μΜ and subjected for indirect detection of various markers. Briefly, samples will be fixed in 4% PFA, permeabilized and blocked in 0.1% BSA, 5% normal goat or donkey serum. Primary antibodies will be diluted accordingly and incubated overnight at 4°C. Alexa Fluor 488 or 594- conjugated secondary antibodies will be used for primary antibodies visualization. Validation of selected markers using primary cancer specimens will narrow the list of candidate genes for creation in future therapies that efficiently target invasive cells.

EXAMPLE 13: Use of Three Dimensional Tumor Spheroid-based Functional Assays to Test Cancer Cell Invasion.

[00100] The preclinical validation process in cancer drug discovery generally comprises a series of primary biochemical and cell-based assays, followed by evaluation in animal tumor models. However, there is a high rate of attrition and fewer than 10% of candidates identified by high throughput screening become licensed drugs. Standard two-dimensional cell cultures for target testing are simple and convenient, but present significant limitations in reproducing the complexity and patho-physiology of in vivo tumor tissue. Three- dimensional culture systems are more representative in cancer research since tissue architecture and the extracellular matrix (ECM) significantly influence tumor cell responses to micro-environmental signals. For functional validation of selected genes as described above, three dimensional tumor spheroid-based functional assays will be used to test which of the selected genes are truly crucial for cancer cell invasion. [00101] Two independent shRNA clones in pTRIPZ lentiviral plasmid (Open Biosystems) will be prepared for each selected gene to be tested. The cell lines will be initially tested by RT- PCR and WB for efficient KD. After validation the 3D system described in Example 9 will be used to test the importance of those genes in invasion. For each clone 20 spheres will be photographed and number and distance of invading cells into the ECM will be counted. In addition to expressing TurboRFP which is expressed from pTRIPZ the cells will be also transduced with baculovirus expressing actin-GFP to visualize actin dynamics. It will allow observe how the KD of particular gene affects cells motility during invasion.

[00102] SCC cell lines deficient in selected genes will show defects in 3D invasion. This functional test combined with data obtained using primary human cancer specimens will indicate potential new markers of invasion and novel targets for anti-metastasis therapeutics.

EXAMPLE 14: Three Dimensional Tumor Spheroid-based Functional Assays

[00103] We established three dimensional tumor spheroid-based functional assays, which allow us to test the role of particular genes in tumor invasion in 3D. As an example we tested the effect of catulin deficiency on invasion in 3D, which we know plays important role in invasion in vivo (Fig. 14). Inducible shRNA lentiviral system was used to generate stable hSCC cell lines that were catulin-deficient upon doxycycline induction (catulin knockdown KD) and scrambled control. Lentivirus transduced catulin control and catulin KD human SCC cells also expressed turboRFP for cell tracking. Those cells were cultured in sphere inducing media (DMEM/F12, N2, insulin, FGF 20 ng/mL and EGF 20 ng/mL) (Fig. 15A and Fig. 15D). In addition those cells can be efficiently transduced with different fluorescently tagged cytoskeletai proteins like actin-GFP for live imaging using baculovirus (Invitrogene). Spheres were then placed into the mixture of collagene and matrigel, and after 48 hours confocal microscopy was performed (Fig. 15B, C-C", catulin control, Fig. 15E, F-F', catulin KD). Note invasive cells marked by arrows in (Fig. 15B and Fig. 15C) in catulin control tumor cells and lack of invasion into the matrix of catulin deficient tumor cells marked by asterix (Fig. 15E and Fig. 15F).

[00104] Sequences listed in this application include the following:

[00105] SEQ ID NO: 1 is the Homo sapiens catulin promoter sequence (1470 bp). ataggtaaggaagtggtgagatggttggctgagaagaattggaagaaaaagttggattga ggac ctgtcctttttgaatgaagaaaaagaagctgaagcttggctatcttcaataccaaagaca gctg tgcagcactggtagattgatgaagctgaagctacaggttgaagcactgtgaagcgccttt caac ggtgcttgggaaagagctttcttatgcctcaggttgatgtacccagctcatcaatctggt gtta tggaagaagaaatgatccaggccaaggtgtccgggtggactggccagcctcagattcttt ttta ctctgacaggttctcactggcagatatggtagttgacaaaacattttttcatgcttctca gtct gttgcttaagtctggaattccttcacggcgtgcatactgctgacaacctccttttggcct tgct cctggagaccttagaataggagtcttcagctccccgcccccatcgtggggctctgtgcga gctc agagttgtgcttacagaactgattcttacccttcccctcaagtgagttgtcagaaataca cact tggtgttttcatgcaatgaacagttatgaagaaactgctctgtgcctggcactaggaaaa aact tcgaaaaatagttttaagaagtagtgttgaacagtagatgaggctttttcatgtttcttt taaa tttcttttgaataagtctaaacagtagtgtgaaagatatactgacttcctgccgaatacc caga tactccattaatgattgcattatcaaatgtttccccttcaatccagctcaaagtgtttgt gaga aatacatggtgaaagaatgagcagggtggagtggaaattggaccttccggagaagcccca ggaa gacattaccactggaattcccgtcccaggggaaaggcccctcccaacagacttgattctg gttt tggtctaccagtttcaaccccagcacagcttccagaagtctttgcgggaattcagagctg attt aggacgagtcacctatcgagaggacgggaagctttgctggggacgcttgtctgtccaagt aaga ggaggccatgtggctgtgtgacagatgtgttttgtttctcctcgccaccctggcgagaag gtgc ctggaacccatggaaggggtctgagaagagataagggcggggccatgtgggggcgggacc cggc ttagggggtggggttgcagggagcccacaccaggctggactgcgcgggcggggcagaggc cggg ggtgggcggggacatacggtgacgggcgtcggggacgcagggttggcggcggagccctgg gggg gcgggcccctgggggcggggtctcggttggcgcgagtgtcctgtcgccgccgcctcgggc gggt gggctgactggcggcaggctcgccgcggcgcggagtcccggctgcgggatagaccgaggg cc

(SEQ ID NO: 1)

[00106] SEQ ID NO: 2 is the polynucleotide sequence encoding the Homo sapiens catulin polypeptide (SEQ ID NO: 3).

1 cgccgcggcg cggagtcccg gctgcgggat agaccgaggg ccatggccgc ctctcccgga

61 cccgccggcg ttggcggcgc cggagcagtc tacggctccg gctcttcggg cttcgccctc

121 gactcgggac tggagatcaa aactcgctcg gtggagcaga cgctactccc gctggtttct

181 cagatcacca cgcttattaa tcataaagat aataccaaaa agtctgataa aactctgcaa

241 gcaattcagc gtgtaggaca agctgtcaac ttggcagttg gaagatttgt taaagtagga

301 gaagctatag ccaatgaaaa ctgggatttg aaagaagaaa taaatattgc ttgtattgaa

361 gctaaacaag caggagaaac aattgcagca cttacagaca taaccaactt gaaccatctg

421 gaatctgatg ggcagatcac aatttttaca gacaaaacag gagtgataaa ggctgcaaga 481 ttacttcttt cttcagtgac aaaagtgttg ttgctggcag accgagtagt cattaaacag 541 ataataacat caagaaataa ggttctcgca actatggaaa gactagagaa agtgaatagc 601 tttcaagagt ttgtccaaat attcagtcaa tttggaaatg aaatggtgga gtttgcacat 661 ctgagtggag atagacaaaa tgatttgaaa gatgaaaaga aaaaggcaaa aatggcagca 721 gctagggcag ttcttgaaaa gtgtacaatg atgcttctca cagcttcaaa gacatgtctg 781 aggcatccta actgcgaatc agcccataaa aacaaagaag gagtatttga ccgtatgaaa 841 gtggcattgg ataaggtcat tgaaattgtg actgactgta aaccgaatgg agagactgac 901 atttcatcta tcagtatttt tactggaatt aaggaattca agatgaatat tgaagctctt 961 cgggagaatc tttattttca gtccaaagag aacctttctg tgacattgga agtcatcttg 1021 gagcgtatgg aggactttac tgattctgcc tacaccagcc atgagcacag agaacgcatc 1081 ttggaactgt caactcaggc gagaatggaa ctgcagcagt taatttctgt gtggattcaa 1141 gctcaaagca agaaaacaaa aagcatcgct gaagaactgg aactcagtat tttgaaaatc 1201 agtcacagtc ttaatgaact taagaaagaa cttcatagta cagcgacaca gctggcagca 1261 gatctattaa aataccatgc tgatcatgtg gttctaaaag cattaaaact tactggagta 1321 gaaggaaatt tagaagcttt ggctgaatat gcctgtaaac tctctgaaca gaaagagcag 1381 cttgttgaga cctgtcgatt gttacgacac atatctggga cagaacctct ggaaataacc 1441 tgtatacatg cagaggagac atttcaggtg actggccaac agataatttc tgctgctgaa 1501 acattgacat tgcatccatc tagtaaaatt gctaaagaaa acctagatgt attttgtgaa 1561 gcttgggaat cccaaattag tgacatgtca acactgctga gagaaatcaa tgacgtgttt 1621 gaaggaagac gaggagagaa gtatggctac ctttcacttc caaagccaat gaagaataat 1681 gcaaacctga aatcattaaa gccagacaag cctgactctg aggagcaagc caagatagca 1741 aagcttggac ttaagctggg tttgctcacc tctgacgctg actgcgaaat tgagaagtgg 1801 gaagatcagg agaatgagat tgttcaatat ggacggaaca tgtccagtat ggcctattct 1861 ctgtatttat ttactagagg agaggggcca ctgaaaactt cccaggattt aattcatcaa 1921 ctagaggttt ttgctgcaga gggtttaaag cttacttcca gtgttcaagc tttttcaaaa 1981 cagctgaaag acgatgacaa gcttatgctt ctcctggaaa taaacaagct aattcctcta 2041 tgccaccagc tccagacagt aactaagact tctttgcaga ataaagtatt tctaaaggtt 2101 gacaagtgta ttacgaagac aagatccatg atggctctct tagtccaact tctttcactt 2161 tgttataaac tgctgaagaa gcttcagatg gaaaataacg gatgggtctc agttacaaat 2221 aaggacacta tggatagtaa aacttgagaa gcttttgggg tcagatctct ggaacatcat 2281 gtgatgaagc tgacattttt aaaaatcaaa tgatccttta tcttttcaga aattcatcaa 2341 ttttataaag aaaacaatat tgaaattttg ctctattttc tgatcatgaa actgattgta 2401 aagctttttg acaactaata aatgtcttgg taattgctag attct (SEQ ID NO: 2)

[00107] SEQ ID NO: 3 is the polypeptide sequence of homo sapiens catenin (cadherin- associated protein), alpha-like 1 (CTNNAL1 ).

MAASPGPAGVGGAGAVYGSGSSGFALDSGLEIKTRSVEQTLLPLVSQITTLINHKDN TKKSDKT LQAIQRVGQAVNLAVGRFVKVGEAIANENWDLKEEINIACIEAKQAGETIAALTDITNLN HLES DGQITIFTDKTGVIKAARLLLSSVTKVLLLADRVVIKQIITSRNKVLATMERLEKVNSFQ EFVQ IFSQFGNEMVEFAHLSGDRQNDLKDEKKKAKMAAARAVLEKCT MLLTASKTCLRHPNCESAHK NKEGVFDRMKVALDKVIEIVTDCKPNGETDISSISIFTGIKEFKMNIEALRENLYFQSKE NLSV TLEVILERMEDFTDSAYTSHEHRERILELSTQARMELQQLISVWIQAQSKKTKSIAEELE LSIL KISHSLNELKKELHSTATQLAADLLKYHADHVVLKALKLTGVEGNLEALAEYACKLSEQK EQLV ETCRLLRHISGTEPLEITCIHAEETFQVTGQQIISAAETLTLHPSSKIAKENLDVFCEAW ESQI SDMSTLLREINDVFEGRRGEKYGYLSLPKPMKNNANLKSLKPDKPDSEEQAKIAKLGLKL GLLT SDADCEIEKWEDQENEIVQYGRNMSSMAYSLYLFTRGEGPLKTSQDLIHQLEVFAAEGLK LTSS VQAFSKQLKDDDKLMLLLEINKLIPLCHQLQTVTKTSLQNKVFLKVDKCITKTRSM ALLVQLL SLCYKLLKKLQMENNGWVSVTNKDTMDSKT (SEQ ID NO: 3)