WO2004086038A2 - A method for screening compounds having the capacity to control tumor cell invasion or metastasis using constitutively active akt - Google Patents

Abstract

The present invention relates to a method for screening compounds having the capacity of controlling tumor cell invasion or metastasis, a method for inhibiting tumor cell invasion, as well as cell lines expressing constitutively active Akt.

Description

A METHOD FOR SCREENING COMPOUNDS HAVING THE CAPACITY TO CONTROL TUMOR CELL INVASION OR METASTASIS USING CONSTITUTIVELY ACTIVE AKT.

BACKGROUND

1. Field of the Invention

The present invention relates to a method for screening compounds having the capacity of controlling tumor cell invasion or metastasis, a method for inhibiting tumor cell invasion, as well as cell lines expressing constitutively active Akt. 2. Prior Art

Most cancer deaths are due to the spread of tumor cells resistant to conventional therapies (1). Metastatic cells acquire genetic and epigenetic changes that cause their aggressive phenotype. The acquisition of increased motility and invasiveness is essential for metastasis (1 ). Increased motility and invasiveness are associated with decreased cell-cell adhesion, degradation of basement membranes and stroma, and enhanced local growth of tumor cells. Some of these properties of metastatic cells have been linked to downregulation of E-cadherin (2, 3), a frequently observed phenotypic change usually caused by transcriptional repression (4-6), and to degradation of basement membranes that is initiated by upregulation of matrix metalloproteinases and collagenases (7).

The oncogenic serine/threonine kinase AKT, also known as Protein Kinase B (PKBα) because of its homology with Protein Kinase A and Protein Kinase C family members, has an amino-terminal pleckstrin homology (PH) domain that binds to the lipid products of phosphoinositide 3-kinase, phosphatidylinositoal-3,4-biphosphate and phosphatidylinositol-3,4,5-triphosphate. Hence, AKT is a downstream effector of the phosphatidylinositol 3' kinase (PI3K) and is frequently activated in human cancer (8). Mammals have at least three distinct genes for AKT family members called AKT1 , AKT2 and AKT3, which are at least partially redundant in function. The gene for AKT2 is amplified and overexpressed in ovarian, pancreatic, breast and follicular thyroid carcinomas, and AKT2 kinase activity is high in ovarian cancer (9-13). Furthermore, total AKT kinase activity is activated in non-small cell lung cancer, squamous cell carcinomas of the oral cavity, breast and prostate carcinomas (14- 16). In ovarian cancer, AKT2 amplification and overexpression are associated with undifferentiated histology and aggressive clinical behavior, suggesting that AKT contributes to tumor progression (10).

AKT activation contributes to the neoplastic phenotype. AKT stabilizes the cell cycle inhibitors p21^Cιp1 and p27^Kιp1 and inhibits the transport of both proteins into the nucleus. AKT also enhances the translation of mRNAs for cyclins D1 and D3. These changes lead to increased cyclin-dependent kinase and E2F activity and promote cell cycle progression (reviewed in 8). AKT also promotes cell survival. The antiapoptotic function of AKT has been linked to inhibition of cytochrome c release from mitochondria, stimulation of glucose uptake and utilization, phosphorylation and inactivation of Bad and (pro)caspase 9, activation of NF-κB, overexpression of Bcl-2 or BclxL, and phosphorylation and nuclear exclusion of FKHRL (8, 17). AKT activation is also associated with enhanced tumor cell invasion. AKT enhances invasiveness of pancreatic carcinoma cells via upregulation of insulin-like growth factor 1 (IGF1 ) (18), and increases secretion of matrix metalloproteinases 2 and 9 from immortalized mammary epithelial cells and ovarian carcinomas (19, 20).

Thus, the present invention has its basis in the investigation of the role of AKT in the biology of human squamous cell carcinoma lines and illustrates that AKT activation causes epithelial-mesenchymal transition (EMT) characterized by downregulation of numerous epithelial cell-specific proteins, including E-cadherin and β-catenin, and upregulation of the mesenchymal cell-specific protein vimentin. Interestingly, EMT was accompanied by increased in vivo cell motility on fibronectin- coated surfaces and increased invasiveness in animals.

Moreover, the present invention reveals that constitutively active Akt can trigger the activation of the Snail, ZEB1 and ZEB 2 promoters, which in turn represses the E. cadherin promoter and thus induces EMT. Thus, Snail, ZEB1 and ZEB2 are new sites that can be targeted for inhibition of tumoral invasion and metastasis by providing compounds that can inhibit activation of the Snail, ZEB1 and ZEB 2. . These findings expand the spectrum of biological activities of AKT and suggest that therapeutic inhibition of AKT is a useful strategy to control tumor cell invasion and metastasis.

The present invention provides a method for screening compounds that inhibit epithelial-mesenchymal transition. In another aspect the present invention provides a method of screening and identifying compounds having the capacity to inhibit or control tumor cell invasion and/or metastasis. Cell lines expressing constitutively active Akt and animal models expressing constitutively active Akt are also provided.

Summary of the Invention In one aspect the present invention provides a method for screening compounds that inhibit epithelial-mesenchymal transition said method comprising:

(a) providing a cell line expressing constitutively active Akt;

(b) contacting said cell line with the compounds to be screened;

(c) measuring at least one biological parameter characteristic of epithelial- mesenchymal transition; and

(d) selecting the compound that controls or inhibits epithelial-mesenchymal transition.

In another aspect, the present invention relates to a method of screening compounds having the capacity to control or inhibit tumor cell invasion or metastasis, said method comprising:

(a) providing a cell line expressing constitutively active Akt;

(b) contacting said cell line with the compounds to be screened;

(c) measuring at least one biological parameter characteristic of tumor proliferation or metastasis; and (d) selecting the compound that controls or inhibits tumor invasion or metastasis.

In yet another aspect, the present invention relates to a method of screening compounds having the capacity to control or inhibit tumor cell invasion or metastasis, said method comprising: (a) providing an animal model that expresses constitutively active Akt;

(b) contacting said animal model with the compounds to be screened;

(c) measuring at least one biological parameter characteristic of tumor proliferation or metastasis; and (d) selecting the compound that controls or inhibits tumor invasion or metastasis. In yet another aspect, the present invention provides a method of identifying a compound that inhibits the transcriptional factors of Snail, ZEB1 and ZEB2, said method comprising: (a) providing a cell line expressing constitutively active Akt;

(b) contacting said cell line with the compounds to be screened;

(c) measuring whether said compound inhibits the transcriptional factors of Snail, ZEB1 and ZEB2; and

(d) selecting compounds that inhibit or preventtumor cell invasion or metastasis.

The biological parameters for use in the above method are selected from at least one of the following methods: a cell migration and attachment test to a substratum, the measurement of down-regulation of beta-catenin and E-cadherin in said cells or animal models, the capacity of said compounds to suppress or decrease tumor invasiveness of said cells into athymic nude mice or a similar mouse model, the loss of epithelial morphology and the assumption of a fibroblast-like appearance in the cell morphology, a change in cell morphology from an epithelial to a mesenchymal appearance, alteration of the subcellular localization of β-catenin and E-cadherin and the capacity of the compound to inhibit the expression of Snail, ZEB1 and ZEB2, which will in turn upregulate E-cadherin expression and prevent epithelial to mesenchymal transition.

In another aspect the present invention provides a cell line expressing constitutively active Akt. In this regard, the cell line is a carcinoma cell line or a metastatic cell line. In yet another aspect the present invention provides a method for inhibiting tumor cell invasion and metastasis comprising administering to a patient in need of such treatment a pharmaceutically acceptable amount of an inhibitor of Akt, alone or in a combination with a conventional treatment.

Brief Description of the Drawings

Fig. 1A are photographs from a phase contrast microscopy experiment showing that Akt affects the morphology of epithelial cells and induces loss of cell- cell adhesion after transient (A - I) or stable (J - M) expression. Morphology of SCC15 cells after infection with an adenovirus expressing either the β-galactosidase marker (A,D,G), a kinase-dead form of Akt (T308A) (B,E,H), or a constitutively active form of Akt, myr-Akt (C,F,I). Cells were either fixed and stained with X-gal (A-C) or directly observed by phase contrast microscopy (D-l). Phase contrast microscopy of G418-resistant SCC15 cells infected with retrovirus expressing v-Akt (L), empty vector (K), or parental cells (J). Note the loss of the epithelial shape and paucity of cell-cell contacts in cells expressing active Akt (F,I,L). Scale bars represent 10 μm (A-C), 40 μm (D-F), and 20 μm (G-L).

Fig. 1 B is a Western blot analysis confirmed the production of viral (v-Akt) and cellular (c-Akt) forms of Akt (upper panel) in SCC15 cells (-), in one c-Akt cell line called (A) and in three independent v-Akt cell lines called (A,C,D). β-tubulin (middle panel) is a loading control. Western blotting with anti-phospho-AKT T308 antibody showed that unlike endogenous AKT, exogenous Akt is active (lower panel).

Fig. 2 are photographs from an immunofluorescence experiment illustrating that Akt induces cellular changes associated with EMT in SCC15 cells. Parental SCC15 cells (A, D), SCC15 v-Akt B (B, E) and SCC15 v-Akt D (C, F) were fixed and processed for immunofluorescence with antibodies recognizing desmoplakin (A-C) and vimentin (D-F). Scale bar: 10 μm.

Fig. 3 shows that Akt downregulates adherent junction and epithelial markers and induces the relocation of E-cadherin and β-catenin. (A) Western blot analysis (E = E-cadherin, α = α-catenin, β = β-catenin, γ = plakoglobin, P = p130cas, H = HEF1) of total lysates of parental SCC15 cells (lane 1 ) and independent mass cultures of SCC15 cell lines infected with retroviruses expressing c-Akt (lane 2), v-Akt (lanes 3,4) or empty vector pLXSN (lanes 5,6). SCC15 parental cells (B,E), SCC15 v-Akt C (C,F) and SCC15 v-Akt D (D,G) were fixed and immunostained with antibodies directed against E-cadherin (B-D) or (β-catenin (E-G); nuclei were labeled with DAPI. Scale bar represents 5 μm.

Fig. 4 shows the activation of SNAIL transcription is associated with repression of E-cadherin transcription in the presence of active Akt. (A) Poly-A Northern blot analysis of the mRNA for β-catenin (CTNB), E-cadherin (CDH1), SNAIL (SNAI1) and GAPDH (GAPD) in SCC15 cells expressing v-Akt (SCC15 v-Akt C) or empty vector (designated 0). The approximate size of the transcripts (in kb) are indicated on the left of the panel. (B) Parental SCC15 (light box) and SCC15 v-Akt C cells (dark box) were co-transfected with pGL3 (-) or pGL3 hE-cad prom (E-cad) as a reporter and with pPGKβgeopA as an internal standard. Two days after transfection, the level of reporter gene transcription was measured as the ratio of luciferase activity to β- galactosidase activity (luciferase/β-gal) and is expressed in relative units (r.u.).

Fig. 5 shows that Akt affects cell migration and attachment to substratum. Figure 5A shows that random cell migration is increased in SCC15 cells expressing v-Akt. SCC15 cells expressing or not expressing exogenous Akt were allowed to attach onto plates coated with 10 μg/ml fibronectin. Cell motility was evaluated by tracking at least 20 cells. The mean and standard error of three independent experiments are shown. Data from migration assays of SCC15, SCC15 c-Akt A, SCC15 v-Akt C, and SCC15 v-Akt D cell lines (ANOVA ; Fp.ee] =53.39, P<0.0001) reveal a statically significant increase in migration induced by v-Akt compared to endogenous or c-Akt (SCC15 versus SCC15 v-Akt C ; t [41]=8.7 ; *** P < 0.0001 , and SCC15 versus SCC15 v-Akt D ; t [44]=9.1 ; ^*** P < 0.0001). Migration induced by v-Akt is significantly different from all other cases (Fisher's analysis : *** P< 0.0001) . Figure 5B shows the result of an experiment concerning the strength of attachment to substratum, which was estimated by the rate of detachment following trypsinization. Solid squares correspond to SCC15 cells, open circles to SCC15 v- Akt C cells, and solid triangles to SCC15 v-Akt D cells.

Fig. 6 shows that Akt induces cell proliferation, tumorigenicity and invasiveness. Figure 6A shows the doubling times for SCC15 cells expressing and not expressing exogenous Akt were estimated from growth curves: 23 h for SCC15 cells (solid squares), 13 h for SCC15 v-Akt B cells (hollow circles), and 12 h for SCC15 v-Akt D cells (solid triangles). Figure 6B shows the oncogenic potential of squamous cell carcinoma lines. The percentage of tumor incidence in nude mice of SCC13 cells infected with retroviruses expressing v-Akt (v), myr-Akt (myr) or empty vector pLSN (0) is shown in the bar graph. The number of tumors per animal injected is indicated for each cell line. (C-F) In vivo invasion assay. Micrographs of tracheal transplant cross sections, stained with hematoxylin and eosin, showing the representative growth pattern of SCC15 cells (D), SCC15 v-Akt B cells (E) and SCC15 v-Akt C cells (F). Cells were initially placed in the lumen (Lu) of the trachea that is surrounded by the cartilage (Car) and the pars membranacea. The v-Akt- expressing cells invaded the tracheal wall and grew in the direction of the arrow after crossing the pars membranacea (D,E). Scale bars: 0.22 mm. (C) The extent of invasion in tracheal transplants (parental SCC15 [-], and SCC15 v-Akt B and C [B and C]) was measured (in mm) and is shown in the histogram along with standard deviations. The number of tumors analyzed (11 to 26) is indicated on the right.

Figure 7 are graphs showing the activation of expression of Snail, ZEB1 and ZEB2 in the presence of active AKT. Figure 7A is a graph showing the real-time semi- quantitative analysis of the level of Snail mRNA in cells expressing c-Akt, v-Akt or empty vector LX. Figure 7B is a graph showing the real-time semi-quantitative analysis of the level of ZEB1 mRNA in cells expressing c-Akt, v-Akt or empty vector LX. Figure 7C is a graph showing the real-time semi-quantitative analysis of the level of ZEB2 mRNA in cells expressing c-Akt, v-Akt or empty vector LX. Figure 7D is a graph showing mock SCC15 LX-B and SCC15 v-AktC cells cotransfected with pGL h Snail 588 luc as a reporter and with PGKβgeopA as an internal control. Figure 7E is a graph showing mock SCC15 LX-B cells cotransfected with pGL h Snail 588 luc as a reporter, 0 or 100 ng of pHT-myr-Akt and with PGKβgeopA as an internal control. Figure 7F is a graph showing mock SCC15 LX-B and SCC15 v-AktC cells cotransfected with pGLSip 195-209 prom 8 as a reporter and with PGKβgeopA as an internal control. Figure 7G is a graph showing mock SCC15 LX-B cells cotransfected with pGLSip 195-209 prom 8 as a reporter, 0 or 100 ng of pHT-myr- Akt and with PGKβgeopA as an internal control.

Preferred Embodiments of the Invention

As used herein, the term "AKT" includes the isomers of AKT1 , AKT2 and AKT3.

As used herein VAkt" is the viral oncoprotein resulting from the fusion of c-akt and a retroviral Gag protein with the inclusion of an additional 21 amino acids derived from the translation of 63 nucleotides of the c-akt 5' untranslated region placed between Gag and Akt.

As used herein, "c-AKT" is the cellular homologue of v-Akt and is also referred to as protein kinase B (PKB).

"EMT" means the epithelial to mesenchymal transition and is characterized by alterations in cell-cell adhesion, cell-substrate interaction, extracellular matrix degradation and cytoskeleton organization.

As used herein "Akt T308A" is a Akt that is a pleckstin homology (PH) domain mutant lacking the threonine at position 308 of the wild-type Akt and therefore lacks kinase activity and cannot be activated by phosphorylation.

"Myr-Akt" is a myristylated Akt which contains all or part of the src mynstoylations signal sequence that permits Akt to translocate to the plasma membrane.

By "constitutively active" is meant that Akt is produced in an excess and constant amount. The term "fibroblast-like properties" means that the cells have a morphological appearance of fibroblast cells which have a stellate or spindle-shape appearance and are capable of forming collagen.

As used herein, "metastasis" means the appearance of neoplasms in parts of the body remote from the site of the primary tumor.

"Invasiveness" denotes the local spread of a malignant neoplasm by infiltration or destruction of adjacent tissue.

The terminology to "control tumor cell invasion" means to inhibit or prevent tumors from forming. Thus the terms, control, inhibit and prevent are used interchangeably herein.

The term "compound" includes any chemical, biological or vegetal substance including organic compounds, lipids, antisense RNA, siRNA, oligonucleotides, deoxyribonucleotides, antibodies, and the like.

The present invention thus relates to squamous cell carcinoma lines engineered to express constitutively-active Akt underwent EMT, characterized by downregulation of the epithelial markers desmoplakin, E-cadherin and β-catenin and upregulation of the mesenchymal marker vimentin. The cells lost epithelial cell morphology and acquired fibroblast-like properties. Additionally, E-cadherin was downregulated transcriptionally. The cells expressing constitutively-active Akt exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals. AKT is activated in many human carcinomas, and the AKT-driven EMT confers the motility required for tissue invasion and metastasis. The inhibition of expression of E-cadherin was linked to the activation of expression of constitutively active AKT and by the consecutive activation of Snail, ZEB1 and ZEB2, thus identifying an additional pathway that can be targeted with compounds to inhibit Snail, ZEB1 and ZEB2, thus upregulating E- cadherin and preventing EMT, indicative of tumor cell invasion and metastasis.

These findings suggest that future therapies based on AKT inhibition may complement conventional treatments by controlling tumor cell invasion and metastasis. Thus, the present invention provides a process for screening and identifying compounds that inhibit the epithelial-mesenchymal transition, which is a cellular process that is associated with development and oncogenesis by which epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility.

In another aspect the present invention relates to a process for screening and identifying compounds having the capacity to control or inhibit tumor cellular invasion and/or metastasis.

In both of the above processes either a cell line containing a constitutively active Akt or an animal model in which tumor cell lines containing a constitutively active Akt are injected, are used in the processes to screen compounds that either inhibit epithelial-mesenchymal transition or control or prevent tumor invasion and/or metastasis. Specific biological parameters are measured in the screening which are indicative that the compounds being tested can inhibit epithelial-mesenchymal transition or control or prevent tumor invasion and/or metastasis.

The present invention also provides a kit containing cell lines expressing constitutively active Akt and reagents necessary to measure at least one biological parameter indicative of tumor proliferation or metastasis.

The constitutively active Akt that is used in the processes and kit of the present invention can be any form of Akt which can be altered in such a manner that when expressed in a recombinant construct in a cell is forced to translocate to the plasma membranes of the cell. It should be appreciated that constitutively active Akt of mammalian origin can be used. In this regard, if the process is directed to screen compounds for human tumor proliferation or human epithelial to mesenchymal transition, then human constitutively active Akt is used. Likewise if the process is used to screen for compounds for veterinary purposes, then the constitutive Akt is of rat, mouse etc. origin.

As an example of constitutively active Akt can be mentioned all or part of the src myhstoylation signal sequence containing the first seven amino acids of Src fused at its N-terminal to a variant of Akt which lacks the pleckstrin homology (PH) domain (myr-Akt).

In another aspect the constitutively active Akt has the following src myhstoylation signal sequence N-terminally fused to Akt lacking the PH domain: MGSSKSKPKDPSQRR (SEQ ID No.1 ) or part of the above Sequence ID No. 1 , as long as the first seven amino acids, which are required for the association of Src with membranes are maintained.

Another example of a constitutively active Akt is a construct in which a Gag polypeptide of v-Akt is fused in-frame to the 5' untranslated portion of the Akt gene such that all of the Akt coding sequence is retained, including the Akt pleckstrin homology (PH) domain.

Yet another example of a constitutively active Akt is a PH domain Akt mutant such as the double mutant T308D/S473D, in which the threonine at position 308 of the native Akt and the serine at position 473 of the native Akt is replaced by aspartic acid.

It will be appreciated that the sequences of Akt of mammalian origin are known as evidenced by Bellacosa et al Science 254:274-277, and the construct of src myristoylation signal sequence N-terminally fused to Akt lacking the PH domain is also known in the art as evidenced by Bellacosa et al Oncogene 17:313-325 (1998).

The above constitutively active Akt can be produced by methods known in the art, by for example, using PCR and confirming the sequence generated by PCR by DNA sequence analysis.

The constitutively active Akt sequences are then inserted into recombinant vectors. Any viral recombinant vectors known in the art can be used such as herpes simplex virus type 1 (HSV-1 ) vectors, retroviral vectors, vaccinia viral vectors, baculoviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), murine leukemia viral vectors and the like. Examples of the various vectors, which are under the control of various promoters and also have a marker gene are described in U.S. Patent Nos. 6,596,535, 6,692,956, 6,686,200 and 6,379,674; in various publications such as Krisky et al., Gene Therapy 4:1120-1125 (1997); Ascadi et al., Human Molecular Genetics, vol 3, No. 4:579-584 (1994); Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630 (1992); and Hermonat et al., PNAS 20:6466-70 (1984). The viral vectors utilized in the present process need not be specific, but in another aspect the vectors are able to express, besides constitutively active Akt, a marker protein such or (-galactosidase or luciferase or an antibiotic resistant gene such as neomycin, as well as the constitutively active Akt.

The vectors are then amplified in particular cell lines such as HEK293 or NIH3T3 fibroblasts and purified or generated by transfection of an amphotropic packaging cell lines or by transient cotransfection into, for example COS cells with an amphotropic packaging plasmid.

The recombinant viral vectors containing the constitutively active Akt are then used to infect various cancer lines. Any cancer cell line can be infected by the recombinant viral constructs set forth above such as MCF7, ZR-75T, MT2994, MCT/18, which are breast carcinoma cell lines, human non-small cell lung cancer (NSCLC) cell lines such as H157, liver cancer cell lines such as HepG2, Hep 3B, HLE and HuH-7, thyroid cancer cell lines such as FTC-133, prostate cancer cell lines such as ALVA-31 , PL-3, DU145, pancreatic cancer cell lines such as PANC-1 , AsPC-1 , BxPC-1 and KP-3, gastric cancer cell lines such as MKN28, MKN45 and MKN74, ovarian cancer cell lines such as Ovca420, Ovca429, Ovca432 andOvca433, human squamous cell carcinoma cell lines such as SCC13 and SCC15, rat carcinoma cell lines such as NBTII, melanoma cell lines such as SK29, SK28 and Mel888, bladder carcinoma cell lines such as T24, VMCubl , VMCub2, 5637, HT1376, SW1710 and SD and the like. It will be appreciated that the cell lines will be selected based on the type of cancer the elected compounds are being tested to screen.

Besides using cell lines, transgenic mammals such as transgenic mice that express constitutively active Akt can also be utilized in the screening processes of the present invention. More specifically, transgenic mice expressing constitutively active Akt can be generated as described by the methods of Shioi et al Embo J. 19:2539-2548 (2000). Generally, these transgenic mice are generated by injecting a cDNA insert encoding constitutively active Akt under the control of a promoter into fertilized mouse eggs and embryos are implanted in the uterus of a surrogate mother. The selected constitutively active Akt will be expressed by some of the offspring. Besides transgenic mice the present invention also includes transgenic rats generated by nuclear transfer as described by Zhou et al., Science, 302: 1179(2003) and AKT transgenic animals generated by CreLox (Sauer, B. Methods, 14:381-92 (1998) or FLP FRT (Cregg et al., Mol Gen Genet, 219:320-3 (1989)) inducing the expression of an active form of AKT. In another aspect, the constitutively active Akt cell lines described above are injected into athymic nude mice. Approximately 1 x 10⁶ to 5 x 10⁶ cells are injected.

Once the cell lines or the animal models described above, are obtained that express constitutively active Akt, the cell line or animal model is incubated with a compound to determine whether the compound inhibits tumor proliferation or metastasis or whether it inhibits epithelial-mesenchymal transition, as indicative by the biological parameters set forth below.

The biological parameters that are used to characterize whether the particular compounds can be selected from those in the art such as cell migration assays, cell- cell adhesion assays, detachment assays, assays for tumorigenesis, invasiveness, change in morphology from epithelial appearance to fibroblast appearance, shifts from epithelial to mesenchymal cell morphology using markers of EMT such as desmoplakin and vimentin alterations in cell adhesion using antibodies against proteins involved in cellular adhesion such as E-cadherin, α-catenin, β-catenin and p130cas, laser scanning confocal microscopy to determine the subcellular localization of E-cadherin, β-cadherin, proliferation and motility of cells, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals and monitoring the activation of expression of Snail, ZEB1 and ZEB2, which in turn inhibits the expression of E-cadherin. The specific details of each of these assays is set forth in the examples below. It is well within the person skilled in this art to adjust the conditions in the disclosed assays, according to the cell line used, as well as the vectors.

In another aspect, the present invention provides a kit which contains the cell line as described above and the reagents necessary to measure at least one of the above-biological parameters.

In order to further illustrate the present invention and advantages thereof, the following specific examples are given, it being understood that the same are intended only as illustrative and in nowise limitative. Examples Materials and Methods

Cell Culture. Squamous carcinoma cell lines SCC13 and SCC15 were derived from human tongue carcinomas (21). Cells were cultured at 37°C and 5% CO2 in DMEM, supplemented with 10% fetal bovine serum.

Antibodies, Immunoblotting and Immunostaining. The mouse monoclonal antibodies used were: anti-p130cas, anti-β-tubulin, anti-desmoplakin l/ll (gifts from Drs. Golemis and Pasdar), anti-vimentin (Biodesign), anti-pan-cadherin (Sigma), anti-human E-cadherin (Transduction Labs) and anti-β-catenin (Transduction Labs). The rabbit polyclonal antibodies used were: anti-HEF1 , anti- -catenin (gifts from Drs. Golemis and Kemler), anti-Akt (19992), anti-phospho-AKT Threonine 308 (Cell Signaling) and anti-γ-catenin (Transduction Labs).

Immunoblotting was as previously described (22). Anti-AKT and anti-HEF1 antibodies were diluted 1/500 before use, anti-p130cas antibody 1/750 and anti-β- tubulin and anti-γ-catenin antibodies 1/2000. Anti-α-catenin, anti-β-catenin, anti-E- cadherin, anti-desmoplakin and anti-pan-cadherin antibodies were used at final concentrations of 2 μg/ml, 0.2 μg/ml, 0.2 μg/ml, 0.5 μg/ml and 15 μg/ml, respectively. Enhanced chemiluminescence detection was used (ECL, Amersham).

Immunostaining was as previously described (22). Antibody concentrations were 2.5 μg/ml for anti-desmoplakin, 0.3 μg/ml for anti-vimentin, 10 μg/ml for anti-E- cadherin and 2.5 μg/ml for anti-β-catenin. Slides were examined under a Leica DM IRB light microscope equipped for epifluorescence or a laser scanning confocal microscope driven by Scanware software (Leica). Images were collected using; the same settings and processed with Adobe Photoshop 4.0 at identical thresholds, to allow semi-quantitative comparisons. Adenoviral Infection. Recombinant adenoviruses encoding β-galactosidase,

Akt T308A or myristylated Akt (myr-Akt) were amplified in HEK293 cells and purified according to standard procedures (23). SCC15 cells were infected with 50 pfu/cell in serum-free medium. After 3 h, infection was stopped by adding medium containing 20% fetal bovine serum. The cells were infected again 2 days later by the same procedure. Phase contrast photographs were taken 2 days later. The proportion of infected cells was estimated from the number of cells producing β-galactosidase, as revealed by X-gal staining.

Retroviral Infection. Inserts harboring c-Akt, v-Akt or myr-Akt were cloned into the retroviral vectors pLXSN (24) or MSV-SRα (25). Infectious viral supematants were generated by transfection of the amphotropic packaging cell line PA-137 for pLXSN-based constructs, and by transient co-transfection of COS cells with the amphotropic packaging plasmid pSV-A-MLV (gift of Dr. Landau) for the MSV-SRD- based constructs. Retroviral infections involved treating subconfluent cultures of SCC13 and SCC15 with DEAE dextran (40 μg/ml) for 1 h and then with viral supematants overnight. G418 (400 μg/ml) was used for selection 48 h after infection, and resistant colonies were pooled. Several mass cultures from independent infections were generated.

Northern Blot Analysis. Total and polyA-RNA were isolated with Roti-Quick and Quick Prep™ kits, respectively. Samples (20 μg of total RNA or 4 μg of polyA- RNA) were subjected to electrophoresis in 1% agarose formaldehyde gels, transferred onto Hybond-N+ membranes and hybridized with ³²P-labeled probes, as indicated. Signals were quantified with Storm 820 using Image Quant 5.2.

Promoter Reporter Assays. SCC15 and SCC15 v-Akt C cells were transiently transfected with Exgen 500 (Euromedex) in 6-well plates. Each well contained serum-free medium containing 1.5 μg pPGKβgeopA and either 2 μg pGL3 basic vector or pGL3 hE-cad prom. Both constructs contain the luciferase gene under no promoter (pGL3) or under the wild-type human E-cadherin promoter (pGL3 hE-cad prom) (6). Luciferase was assayed by standard procedures and transfection efficiency was normalized for β-galactosidase activity.

Cell Migration Assay. Freshly trypsinized cells were plated at 10⁵ cells on 35- mm Falcon 3004 dishes, coated with 10 μg/ml fibronectin, 25 μg/ml collagen or 50 μg/ml laminin. The average speed (μm/h) of locomotion in complete medium, for optimal migration (26), was calculated for 20-30 cells for each experimental condition.

Detachment Assay. Cells were seeded at 1.5 x 10⁵ in 25 cm² flasks. After 48 h, cells were washed with 5 ml of warm PBS, and then trypsinized with 1 ml of fresh 0.25% trypsin (Gibco-BRL # 25300-054) at 20°C with gentle agitation. The number of detached cells was determined at various times and the total number of cells per flask was determined after complete trypsinization. One flask was used for each time point and each experiment was performed at least five times independently.

Growth Curve. Cells were seeded at 1 x 10⁵ in 25 cm² flasks, fed every other day and counted every day. Growth curves were constructed, and doubling times estimated. Assays for Tumorigenesis and Invasiveness. To determine tumorigenicity,

SCC13 cells (5 x 10⁶) were transferred subcutaneously into athymic nude mice. Eight weeks after injection, mice were killed and examined for gross evidence of tumors. Tissues were collected from the injection area, stained with hematoxylin/eosin and analyzed. A tracheal invasion assay was performed as previously described (21 , 27, 28):

5 x 10⁵ cells were injected into the lumen of de-epithelialized rat tracheas that were closed with metal clips at both ends and then transplanted subcutaneously into nude mice. After 4 or 8 weeks, transplanted tracheas were removed and processed for histopathology. Invasiveness was estimated as the extent of penetration into the tracheal wall from the center of the cell mass to the most distal invasive point (28). Results

Constitutively Active Akt Alters the Morphology of Squamous Cell Carcinoma Lines. The human squamous cell carcinoma line SCC15 was infected with recombinant adenoviruses expressing β-galactosidase, Akt T308A (kinase- dead) or myristylated Akt (myr-Akt, constitutively active) (29). The titer of all three adenoviruses was similar. X-gal staining showed that about 45% of the cells exposed to the β-galactosidase adenovirus were actually infected (Fig. 1A). β- galactosidase- or Akt T308A-infected cells displayed no morphological changes (Fig. 1A,B,D,E,G,H). Cells infected with myr-Akt lost their epithelial cell morphology; they were dispersed and assumed a fibroblast-like appearance (Fig. 1C,F,I). Similar results were obtained with NBT-II, a rat carcinoma cell line (data not shown).

Similar experiments were performed using empty retroviruses, retroviruses expressing c-Akt or another constitutively active mutant, v-Akt. The v-Akt oncoprotein contains amino-terminal viral Gag sequences that provide a myristylation site (30, 31). Three mass cultures of cells infected with pLXSN c-Akt (called SCC15 c-Akt A, B and C), four mass cultures of cells infected with pLXSN v- Akt (SCC15 v-Akt A, B, C and D) and two of cells infected with empty retrovirus (SCC15-pLXSN A and B) were subjected to G418 selection. Lysates from uninfected and infected cultures were analyzed by immunoblotting using an antibody that recognizes both endogenous human AKT and exogenous murine Akt. (-tubulin was used as loading control (Fig. 1 M). Uninfected SCC15 produced the 60 kDa endogenous AKT. SCC15 c-Akt cells contained at least three times more Akt than SCC15 cells. Various amounts of a protein larger than 85 kDa corresponding to v- Akt were detected in SCC15 v-Akt A, C and D cells. AKT activation status was examined by western blotting with anti-phospho-AKT Threonine 308 antibody, a marker of AKT activation. Total AKT activity was very low in parental SCC15 cells and high in SCC15 cells expressing exogenous Akt (Fig. 1M). Expression of constitutively active Akt (v-Akt) was associated with the transition from an epithelial to a fibroblast-like morphology (Fig. 1 J-L). Constitutively Active Akt Promotes a Shift in Expression from an Epithelial to a Mesenchymal Repertoire.

The morphological effect of v-Akt expression on squamous cell carcinoma lines suggested that active Akt promotes an epithelial-mesenchymal transition (EMT). The morphological changes characteristic of cells undergoing EMT are accompanied by a shift in gene expression from an epithelial to a mesenchymal repertoire. To determine whether Akt promotes such a shift, we used immunofluorescence to examine the expression and subcellular distribution of desmoplakin and vimentin, two markers of EMT. In parental SCC15 cells, desmoplakin was mostly in spots at the sites of cell-cell contact (Fig. 2A). All v-Akt infected cells contained desmoplakin, but only in cytoplasmic granules (Fig. 2B and C), and less abundant than in SCC15 cells, as assessed by Western blot analysis (data not shown). The parental SCC15 cell line did not produce vimentin, a mesenchymal marker (Fig. 2E, 2D), whereas all v-Akt cell lines produced vimentin microfilaments (Fig. 2E and F). The heights of SCC15, SCC15 v-Akt C and SCC15 v-Akt D cells were determined by confocal microscopy using E-cadherin as marker (data not shown). The mean height of SCC15 cells was 6.0 μm, and the mean heights of SCC15 v-Akt C or SCC15 v-Akt D cells were similar and equal to about 3.8 μm. We conclude that constitutively active Akt indeed induces EMT. Constitutively Active Akt alters the expression of Cell Adhesion

Molecules. The integrity of adherent junctions and other structures involved in cell to cell contact is essential for the maintenance of epithelial structures. To determine whether Akt disrupted such structures, lysates from SCC15 cells expressing active Akt were probed with antibodies against proteins involved in cell adhesion (Fig. 3A). E-cadherin, β-catenin, α-catenin and p130cas were all dramatically downregulated in v-Akt- but not c-Akt- or vector-infected cells. The level of plakoglobin was slightly down-regulated and the level of the p130cas substrate HEF1 (human enhancer of filamentation 1) was not downregulated. N-cadherin was slightly upregulated (data not shown). Similar experiments with SCC13, another human squamous cell carcinoma line, and MSV-SRα retroviruses encoding myr-Akt revealed changes in expression indistinguishable from those in SCC15 v-Akt (data not shown).

Constitutively Active Akt Downregulates E-Cadherin and β-Catenin and Alters their Subcellular Localization. To confirm the immunoblot findings and determine the localization of E-cadherin and β-catenin in the SCC15 cell lines, we used laser scanning confocal microscopy (Fig. 3B-G). Parental SCC15 cells produced both E-cadherin and β-catenin and both proteins were at contact sites between cells (Fig. 3B,E). v-Akt-expressing-SCC15 cells contained less β-catenin and E-cadherin: most of the E-cadherin was in cytoplasmic granules with only a small amount at the plasma membrane (Fig. 3C,D); β-catenin was diffuse in the cytoplasm, although some remained at the membrane (Fig. 3F,G). Slight quantitative differences in β-catenin distribution were observed between SCC15 v-Akt C and SCC15 v-Akt D cells; the amount of β-catenin at the membrane was slightly higher in SCC15 v-Akt C cells. In the cytoplasm of Akt-expressing cells, E-cadherin and β- catenin did not colocalize (Fig. 3F,G and data not shown).

Constitutively Active Akt Downregulates the Expression of the E- Cadherin Gene Transcriptionally by Inducing SNAIL. During EMT there is a massive shift of gene expression from a pattern characteristic of epithelial cells to that of mesenchymal cells. There may be genes that are the primary targets of the EMT-inducing signal(s) and others whose expression is affected secondarily. E- cadherin and β-catenin are potential candidate genes whose downregulation by Akt may be direct, so we examined their mRNAs in v-Akt-expressing and parental SCC15 cells. Akt did not significantly affect the level of β-catenin mRNA, but substantially reduced the level of E-cadherin mRNA (Fig. 4A). Parental SCC15 and v-akt-expressing-SCC15 cells were transiently transfected with pGL3 or pGL3 hE- cadherin promoter luciferase constructs to determine whether Akt modulates the activity of the E-cadherin promoter. Luciferase assays of extracts from the transfected cells revealed that the activity of the E-cadherin promoter was repressed by Akt (Fig. 4B). Similar results were obtained for the murine E-cadherin promoter (data not shown). Thus Akt appears to downregulate E-cadherin gene transcription.

The E-cadherin promoter is a direct target of the transcriptional repressor

SNAIL (32), and we therefore tested whether Akt regulates SNAIL expression or activity. We examined the abundance of SNAIL mRNA in parental and v-Akt- expressing cells, and found that SNAIL is indeed induced by constitutively active Akt

(Fig. 4A).

Constitutively Active Akt Alters Cell Motility on Different Substrates and Cell Attachment to Plastic. Motility of parental and Akt-expressing-SCC15 cells (SCC15, SCC15 c-Akt A, SCC15 v-Akt C and SCC15 v-Akt D cells) was examined on uncoated plastic and plastic that had been coated with fibronectin, collagen or laminin. On fibronectin, SCC15 v-Akt C and v-Akt D cells migrated more rapidly than parental SCC15 cells, whereas SCC15 c-Akt A cells exhibited the same motility as the parental SCC15 cells (Fig. 5A). On laminin, SCC15 v-Akt C cells migrated more slowly than SCC15 cells, whereas on collagen, the speed of migration of all cell lines was similar (data not shown).

The strength of cell anchorage to plastic substratum was also determined. SCC15 v-Akt C and v-Akt D cells detached after 9 min in trypsin, whereas parental SCC15 cells detached after 25 min in trypsin (Fig. 5B). Thus, constitutively active Akt attenuates the attachment of SCC15 cells to plastic.

Constitutively Active Akt promotes proliferation, tumorigenicity and invasiveness of squamous cell carcinoma lines. E-cadherin suppresses tumor invasiveness (3, 33), so it was examined to determine whether Akt potentiates the tumorigenic potential and invasiveness of epithelial cells (Fig. 6). Growth curves were constructed for SCC15, SCC15 v-Akt B and v-Akt D cells (Fig. 6A). The doubling time of the parental cells was 23 h, and those of SCC15 v-Akt B and v-Akt D were only 13 and 12 h, respectively. Similar experiments were performed with SCC13 cell lines transfected with empty vector (Srα), v-Akt and myr-Akt. The respective doubling times were estimated to be 28 hours, 13 hours, and 17 hours for SCC13-Srα, SCC13-v Akt, and SCC13-myr Akt, respectively (data not shown). SCC13 cells are not tumorigenic in athymic nude mice (27). To determine whether Akt is sufficient to render SCC13 cells oncogenic, tumor formation was evaluated by vector-infected- and c-Akt-, v-Akt- or myr-Akt-expressing SCC13 cells. Parental cells, vector- and c-Akt-infected SCC13 cells could not produce tumors in nude mice, whereas SCC13 cells expressing v-Akt and SCC13 myr-Akt cells produced tumors (Fig. 6B). Microscopic examination of these tumors revealed evidence of invasion of the surrounding muscular tissue. Unlike parental SCC13 cells, parental SCC15 cells are fully tumorigenic in nude mice (21) and therefore were not used to score for the effect of constitutive Akt activation on tumorigenesis. The ability of parental and Akt-expressing SCC15 cells to pass through the pars membranacea of tracheal walls to assess invasiveness was measured (Fig. 6C- F). The cells expressing ectopic v-Akt migrated more efficiently through tissues than did the parental SCC15 cells. The test of invasiveness was also performed with the various SCC13 cell lines yielding virtually identical results: SCC13 cell lines expressing v-Akt or myr-Akt, but not SCC13-Srα cells, were able to pass through the pars membranacea (data not shown). Therefore, constitutively active Akt is a potent promoter of tumorigenicity and invasion.

Reverse transcription and real-time semi-quantitative PCR: 1 μg of RNA was reversed transcribed. Real-time semi-quantitative PCR analysis for Snail, ZEB1 and ZES2 cDNAs were performed using the iCycler (BioRad) instrument and software. The sequences of the primers used were as follows:

Snail: 5' TGC AGG ACT CTA ATC CAA GTT TAC C 3' (LL511) (SEQ ID No. 2) and 5' GTG GGA TGG CTG CCA GC 3' (LL512) (SEQ ID No. 3); ZEB 1: 5' CTG CCA ACA GAC CAG ACA GT 3' (LL591) (SEQ ID No. 4)and 5'

AGG ATT TCT TGC CCT TCC TT 3' (LL592) (SEQ ID No. 5); and

ZEB 2 (SIP1): 5' GCG GCA TAT GGT GAC ACA CAA 3' (LL517) (SEQ ID No. 6) and 5' CAT TTG AAC TTG CGA TTA CCT GC 3' (LL518) (SEQ ID No. 7). Promoter reporter assays: SCC15, SCC15 Lx-B and SCC15 v-Akt C cells were transfected with Exgene 500 (Euromedex) in 6-well plates. Each well contained serum-free medium containing 1.5 μg of PGKβgeopA and 2 μg of either pGL3 basic vector or pGL3 hE-cad prom (-306/+21 ) (#624, Comijn et al 2001 ) or pGL3-E-cadh prom (-178/+92) (#772, Battle et al 2000) or pGL enh Sip 306-282 prom2 (#718, containing a 669 bp fragment of the SIP1 distal promoter) or pGLSip 259-249 promoter2(#717, containing a 1081 bp fragment of the SIP1 middle promoter) or pGLSip 195-208 promδ (#719, containing a 482 bp fragment of the SIP1 proximal promoter)or 1 μg of 3-enh-κB-CONA-luc vector (#765, Arenzana-Seisdedos, F. et al, Journal of Cell Science 1997) or 0.5 μg of pGL h Snail 588 luc (#740) containing the -588 to +92 human Snail promoter fragment cloned into the pGL3 basic vector. The luciferase activity of these vectors was determined in the presence of either doses of pcDNA3-mm snail-HA (#502), Nieto et al, Development 116 (1) pgs. 227-237 (1992)) or pCS3-mSIP1 FL (#673, Remade et al, 1999 Embo J. 18: 5073-5084) or pCS3mDeltaEF1 F: (# 674 Remade et al , 1999, Embo J. 18:5073-5084) for either pGL3 hE-cad prom (-308/+21 ) or pGL3-E-cadh prom (-178/+92) or different doses of pcDNA3 Ikappa B S32/36A (#764, Hay, DC, Molecular and Cellular Biology, 2001) for either pGL3 hE-cad prom (-308/+21 ) or pGL3-E-cadh prom (-178/+92) or 3-enh- KB-CONA-IUC vector or pGL h Snail 588 luc or pGLSip 195-209 promδ or different doses of pHT-myr-Akt (#450, Bellacose, A et al, Oncogene 1998) for either pGL h Snail 558 luc or pGLSip 195-209 promδ. Luciferase was assayed using standard procedures and transfection efficiency was normalized for β-galactosidase activity. Signaling pathway downstream Akt leading to the repression of the E-cadherin promoter:

Akt-induced EMT is also associated with the downregulation of E-cadherin protein level. Active Akt represses the activity of the E-cadherin promoter. Transcription factors of the Snail family and the ZEB1 family are known to bind to and repress the E-cadherin promoter. Their ectopic expression induces an EMT. Therefore, the mRNA level of these different factors was determined in SCC15 cell lines (Figure 7 A-C). The expression of Snail, ZEB1 and ZEB2 was upregulated in SCC15 expressing active Akt compared to mock cells. The activity of the Snail and ZEB2 promoters was determined in SCC15 Lx-B and SCC15 v-Akt C cells (Figure 7 D-G). A 58δ bp fragment of the human Snail promoter is 3 times more active in SCC15 v-Akt C cells than in SCC15 Lx-B (Figure 7D). The co-transfection of active Akt with this Snail reporter vector led to its activation within 72 hours following transfection (Figure 7E). Therefore, active Akt can trigger the activation of the Snail promoter in a quite limited number of steps. The ZEB1 gene contains three different promoters termed distal, middle and proximal, The activities of the distal and middle promoters were similar in SCC15 Lx-B and SCC15 v-Akt C cells (data not shown) A 4δ2 bp fragment of the human ZEB2 middle promoter is 9.4 times more active in SCC15 v-Akt C cells than in SCC15 Lx-B (Figure 7F). The co-transfaction of active Akt with this ZEB2 reporter vector led to its activation with 72 hours following transfection (Figure 7G). Therefore, active Akt can trigger the activation of the ZEB2 middle promoter in a quite limited number of steps. Discussion During EMT, epithelial cells acquire fibroblast-like properties and exhibit reduced cell-cell adhesion and increased motility. The plasticity afforded by EMT is central to the complex remodeling of embryo and organ architecture during gastrulation and organogenesis. In pathological processes such as oncogenesis, EMT may endow cancer cells with enhanced motility and invasiveness. Indeed, oncogenic transformation may be associated with signaling pathways promoting EMT (34). AKT activation is frequent in human epithelial cancer (δ-13, 15, 16). Interestingly, in ovarian carcinomas, AKT2 activation has been linked to aggressive clinical behavior and loss of histological features of epithelial differentiation (10). These findings are consistent with AKT directly affecting epithelial cell morphology, tumorigenicity, cell motility, and invasiveness. Here we show that constitutively active Akt induces EMT and stimulates proliferation and motility of squamous cell carcinoma lines plated on fibronectin- coated surfaces (Fig. 5). Also, active Akt promotes invasiveness (Fig. 6). Cells expressing a constitutively active mutant of Akt (v-Akt or myristylated Akt) displayed several features typical of EMT: reduction in cell-to-cell adhesion, and flattening and spreading or scattering (Fig. 1 ). We detected Akt-induced EMT in cells stably infected with retroviral vectors and those transiently infected with adenoviral vectors. EMT occurred a minimum of 72-96 h after transient infection, which may reflect the time required for the reprogramming of gene expression and/or structural reorganization associated with EMT (34). At least two separate correlates of EMT have been identified, namely cell-cell dissociation and cell movement (34). Akt activation appears to mediate both processes. In particular, expression of active mutants of Akt increases cell migration on fibronectin-coated plates, but reduces migration on laminin-coated plates. This pattern is compatible with induction of α4β1 integrin that interacts specifically with fibronectin (35). Indeed, integrin activation often follows EMT (36, 37).

In several cell culture models, EMT is induced by TGFβ or by peptide growth factors via receptor tyrosine kinase signaling (22, 3δ-41). In both cases, PI3K is a critical mediator of EMT. Oncogenic SRC and RAS, both inducers of EMT, also activate PI3K (42). Our data suggest that AKT kinases are major effectors of EMT signals downstream of PI3K. Potential targets of the P13K/AKT pathway include Rac and Rho, two small G proteins involved in cytoskeletal reorganization, cell migration and invasiveness (34).

Akt-induced EMT involves a large downregulation of E-cadherin and β-catenin protein levels (Fig. 3). Downregulation is specific for E-cadherin, as the closely related N-cadherin is not affected. E-cadherin and β-catenin are also relocalized to separate compartments, an indication that their interaction is disrupted. E-cadherin is internalized and displays a punctate cytoplasmic staining pattern, compatible with a vesicular localization. Localization of E-cadherin in vesicles has been described during IGF1 -induced EMT and may point to alterations in protein trafficking, possibly induced by activation of Rab5-mediated endocytosis (22, 43-45).

Akt also downregulated β-catenin. In the Wnt pathway, glycogen synthase kinase 3 (GSK3) phosphorylates axin and β-catenin, causing degradation of the latter. Upon Wnt-induced inhibition of GSK3, β-catenin accumulates in the cytoplasm and translocates into the nucleus acting as a cofactor for the transcription factor LEF/TCF, affecting the transcription of genes that promote cell survival and proliferation (46-50). AKT, activated by peptide growth factor signals, phosphorylates and inhibits GSK3 (51), so we expected stabilization and nuclear translocation of β- catenin in squamous cell carcinoma lines expressing active Akt. The apparent discrepancy can be resolved by recent crystallographic and biochemical studies elucidating the mechanisms of GSK3 regulation (52, 53): Wnt and insulin/AKT signaling pathways affect two distinct pools of GSK3 that in turn target different substrates, thereby giving selective responses and differential substrate phosphorylation (54). In the presence of sustained Wnt signaling, phosphorylation of GSK3 by AKT potentiates the Wnt pathway leading to β-catenin stabilization; however, AKT signaling alone cannot initiate the Wnt signaling process (55-57). Consequently, in our system, activation of Akt alone, i.e., in the absence of Wnt signaling, may not be able to phosphorylate and inhibit the GSK3 pool involved in β- catenin upregulation. This appears to be a post-transcriptional effect. Possibly, β-catenin is destabilized and even degraded as a secondary consequence of the downregulation of E-cadherin. Following Akt activation β-catenin was displaced and did not colocalize with E-cadherin (Fig. 3), and in both Drosophila and mouse development, binding to E-cadherin stabilizes β-catenin (5δ, 59). In addition to intemalization of E-cadherin, Akt activation represses E-cadherin gene transcription (Fig. 4). In the presence of active Akt, the E-cadherin promoter is less active and this repression appears to be the consequence of upregulation of the transcription repressor SNAIL. Indeed, SNAIL induces EMT by repressing E- cadherin transcription (4, 5). Other potential modulators of E-cadherin transcription are SNAIL-related repressors, such as Slug, Smuc, and SIP1 that bind to E-boxes in the E-cadherin promoter (6, 32, 60). The mechanisms by which Akt activates transcription of SNAIL remain unclear.

Akt-induced EMT endows squamous cell carcinoma lines with an invasive phenotype as demonstrated by an in vivo assay of invasion (Fig. 6). The identical assay has demonstrated that AKT2 antisense RNA can inhibit invasiveness in cancer cells that amplify/overexpress the AKT2 gene (11). Although the invasiveness may be in part due to the ability of Akt to stimulate cell cycle progression (Fig. 6), it is more likely to result from loss of cell-cell adhesion (Fig.1-3), increased motility (Fig. 5) and tissue degradation. Akt activation can lead to increased production of matrix metalloproteinases (19, 20) and, in turn, low levels of E-cadherin are associated with stromelysin 1 activation (61). Overexpression of IGF1 R in the pancreas in vivo leads to transformation and invasion associated with downregulation of E-cadherin (62). An intriguing possibility is that this effect of IGFR1 is mediated by AKT.

In summary, it has been demonstrated that activation of the AKT pathway in cancer cells leads to EMT and invasion in vivo. Thus, an important consequence of the AKT activation often detected in human carcinomas is the acquisition of an invasive phenotype. Therapy based on AKT inhibition may therefore complement conventional treatments to control tumor cell invasion and metastasis. Also the first time it has been demonstrated that the down regulation of the expression of E.cadherin by inducing expression of Snail, ZEB1 and ZEB2 is another pathway which can be used to identify compounds that inhibit tumor cell invasion.

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Claims

What is claimed is:

1. A method for screening and identifying compounds that inhibit epithelial- mesenchymal transition said method comprising:

(a) providing a cell line expressing constitutively active Akt;

(b) contacting said cell line with the compounds to be screened;

(c) measuring at least one biological parameter characteristic of epithelial- mesenchymal transition; and (d) selecting the compound that controls or inhibits tumor invasion or metastasis.

2. The method according to Claim 1 , wherein said cell line is a cancer cell line selected from the group of: rat carcinoma cell lines, breast carcinoma cell lines, melanoma cell lines, bladder cancer cell lines, non-small cell lung cancer cell lines, liver cancer cell lines, pancreatic cancer cell lines, gastric cancer cell lines, thyroid cancer cell lines, prostate cancer cell lines, squamous cell carcinoma cell lines. and ovarian cancer cell lines.

3. The method according to Claim 1 , wherein said cell line is a squamous carcinoma cell line.

4. The method according to Claims 1 or 2, wherein said cell line is selected from the group of MCF7, ZR-75T, MT2994, MCT/18, H157, HepG2, Hep3B, HLE, HuH-7, FTC-133 ALVA-31 , PL-3, DU145, PANC-1 , AsPXC-1 , BxPC-1 KP-3, MKN28, MKN45, MKN74, Ovca420, Ovca429, Ovca432, Ovca422, SCC13, SCC15, SK29, SK28, Mel 888 and NBT II.

5. The method according to any one of Claims 1 to 4, wherein said constitutively active Akt is selected from the group of myr-Akt, a src myristoylation signal sequence comprising all of part of SEQ ID No:1 N-terminally fused to Akt lacking the PH domain, a Gag polypeptide of v-Akt is fused in-frame to the 5' untranslated portion of the Akt gene such that all of the Akt coding sequence is retained, including the Akt pleckstrin homology (PH) domain and a PH domain Akt mutant such as the double mutant T308D/S473D.

6. The method according to any one of Claims 1 to 5, wherein said biological parameter indicative of tumor proliferation is selected from the group of: change in morphology from epithelial appearance to fibroblast appearance, shifts from epithelial to mesenchymal cell morphology, a cell migration assay, a cell-cell adhesion assay, a detachment assay, assays for tumorigenesis, invasiveness, alterations in cell adhesion, laser scanning confocal microscopy to determine the subcellular localization of E-cadherin, β-cadherin, proliferation and motility of cells, increased motility on fibronectin-coated surfaces and activation of expression of Snail, ZEB1 and ZEB2.

7. The method according to Claim 6, wherein shifts from epithelia to mesenchymal cell morphology is measured using markers of desmoplakin and vimentin.

8. The method according to Claim 6, wherein said alterations in cell adhesion are measured using antibodies of E-cadherin, β-catenin, α-catenin and p130cas.

9. The method according to any one of Claim 1 to 6, wherein the biological parameter measured is E-cadherin and said compound upregulates E-cadherin, which inhibits or prevents tumor cell invasion.

10. A method for screening and identifying compounds that have the capacity to control or inhibit tumor invasion or metastasis, said method comprising: (a) providing a cell line expressing constitutively active Akt;

(b) contacting said cell line with a compound to be screened;

(c) measuring at least one biological parameter indicative of tumor proliferation or metastasis; and (d) selecting the compound that controls or inhibits tumor invasion or metastasis.

11. The method according to Claim 10, wherein said cell line is a cancer cell line selected from the group of: rat carcinoma cell lines, breast carcinoma cell lines, melanoma cell lines, bladder cancer cell lines, non-small cell lung cancer cell lines, liver cancer cell lines, pancreatic cancer cell lines, gastric cancer cell lines, thyroid cancer cell lines, prostate cancer cell lines, squamous cell carcinoma cell lines and ovarian cancer cell lines.

12. The method according to Claim 10, wherein said cell line is a squamous carcinoma cell line.

13. The method according to Claims 11 or 12, wherein said cell line is selected from the group of MCF7, ZR-75T, MT2994, MCT/18, H157, HepG2, Hep3B, HLE, HuH-7, FTC-133 ALVA-31, PL-3, DU145, PANC-1 , AsPXC-1 , BxPC-1 KP-3, MKN28, MKN45, MKN74, Ovca420, Ovca429, Ovca432, Ovca422, SCC13, SCC15 SK29, SK28, Mel 888 and NBT II.

14. The method according to any one of Claims 10 to 13, wherein said constitutively active Akt is selected from the group of myr-Akt, a src myristoylation signal sequence comprising all of part of SEQ ID No:1 N-terminally fused to Akt lacking the PH domain, a Gag polypeptide of v-Akt is fused in-frame to the 5' untranslated portion of the Akt gene such that all of the Akt coding sequence is retained, including the Akt pleckstrin homology (PH) domain and a PH domain Akt mutant such as the double mutant T308D/S473D.

15. The method according to any one of Claims 10 to 14, wherein said biological parameter indicative of tumor proliferation is selected from the group of: change in morphology from epithelial appearance to fibroblast appearance, shifts from epithelial to mesenchymal cell morphology, a cell migration assays, a cell-cell adhesion assay, a detachment assay, assays for tumorigenesis, invasiveness, alterations in cell adhesion, laser scanning confocal microscopy to determine the subcellular localization of E-cadherin, β-cadherin, proliferation and motility of cells, increased motility on fibronectin-coated surfaces and activation of expression of Snail, ZEB1 and ZEB2.

16. The method according to Claim 15, wherein shifts from epithelia to mesenchymal cell morphology is measured using markers of desmoplakin and vimentin.

17. The method according to Claim 15, wherein said alterations in cell adhesion are measured using antibodies of E-cadherin, β-catenin α-catenin and p130cas.

18. The method according to any one of Claims 10 to 17, wherein the biological parameter measured is E-cadherin and said compound upregulates E-cadherin, which inhibits or prevents tumor cell invasion or metastasis.

19. A method of identifying a compound that inhibits the transcriptional factors of Snail, ZEB1 and ZEB2, said method comprising: (a) providing a cell line expressing constitutively active Akt;

(b) contacting said cell line with the compounds to be screened;

(c) measuring whether said compound inhibits the transcriptional factors of Snail, ZEB1 and ZEB2; and

(d) selecting compounds that inhibit or prevent tumor cell invasion or metastasis.

20. A method of screening compounds having the capacity to control or inhibit tumor cell invasion or metastasis, said method comprising:

(a) providing an animal model that expresses constitutively active Akt;

(b) contacting said animal model with the compounds to be screened;

(c) measuring at least one biological parameter characteristic of tumor proliferation or metastasis; and

(d) selecting the compound that controls or inhibits tumor invasion or metastasis.

21. The method according to Claim 20, wherein said animal model is a transgenic mouse or a transgenic rat.

22. The method according to Claim 20, wherein said animal model is a athymic nude mouse.

23. A kit comprising a cell line expressing constitutively active Akt and reagents necessary to measure the biological parameters of tumor invasion or metastasis.