This invention relates to the field of treatments for cancer.
The receptor tyrosine kinase (RTK), Met, also known as hepatocyte growth factor receptor is involved in diverse cellular functions such as scattering, invasion, proliferation, morphogenesis and angiogenesis (1). It is also implicated in a large number of human tumours, correlating closely with metastasis and poor prognosis (2, 3). These tumorigenic activities occur when Met is overexpressed, or when Met signalling after ligand (HGF) contact is not downregulated (4). Several polypeptide fragments and variants have been found to be activators of Met. See US 2004/0236073, published Nov. 25, 2004; European Patent EP 0 981 620, published Feb. 25, 2004, which are herein incorporated by reference. In contrast, cells can stop signalling downstream of Met, as well as other RTKs, through ligand-dependent endocytosis and subsequent degradation of activated receptor (4-6).
Ubiquitination is a post-translational modification of proteins in which the modifier is itself a polypeptide that is conjugated to the target proteins by forming an isopeptide bond between the carboxyl terminus of ubiquitin and a lysine side chain(s) in the target protein (7, 8). Protein modification by ubiquitin occurs in three successive steps mediated by the enzymes E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ubiquitin ligase). Ubiquitin molecules can form poly-ubiquitin chains conjugated to target proteins that are normally recognized and degraded by the proteasome (7, 9). However, covalent attachment of only one ubiquitin module (mono-ubiquitin) or multiple mono-ubiquitin modules does not result in proteasomal degradation of modified protein, but has been shown to modulate biological functions such as endocytosis and transport to lysosomes (7, 10, 11). Recent studies have shown that ligand-dependent endocytosis of RTKs and other membrane proteins is triggered by mono-ubiquitination of the receptor, mediated by the ubiquitin ligase Cbl (6, 12-18). This picture is more complicated in the case of Met, whose mechanism of degradation after endocytosis is unclear because both proteasomal (19) or lysosomal (20) pathways have been described as degradation mechanisms. Furthermore, Met-ubiquitination has been reported to be monoubiquitination (21) or polyubiquitination (15, 20), even though Cbl is the ubiquitin ligase that recognizes Met (13, 15, 22).
L. monocytogenes is an intracellular pathogen able to use Met as receptor to promote its internalization into normally non-phagocytic epithelial cells (23-25). The bacterial protein that interacts with Met is a protein of the internalin family called In1B (25-28). The interaction between In1B and Met triggers the tyrosine phosphorylation of Met and the recruitment to activated Met of different proteins e.g. Gab1, Shc, and Cbl, among others (29-31). Soluble In1B behaves as a growth factor i.e. triggering similar signalling events and membrane ruffles (30). Remarkably, Cbl is recruited during the first minute following In1B addition to cultured cells and Met becomes rapidly phosphorylated (31). Moreover, latex beads coated with In1B are also able to enter into cells, indicating that In1B is sufficient to promote phagocytosis (24, 32).
Agents that inhibit Met signalling represent an important therapeutic avenue for the treatment of a variety of malignant tumors. For example, antibiotics of the Geldamycin family that down-regulate the expression of Met are in clinical trials for cancer treatments (55, 56). In addition, the anticancer activities of antagonistic antibodies against Met and neutralizing antibodies against Met ligand (HGF) are also important potential anticancer therapies. There is a need in the art for new treatments for cancer based on the activities of Met.
The invention fulfills this need by illuminating a pathway by which Met is degraded after interacting with In1B, in response to Listeria monocytogenes infection. Listeria uses the surface protein In1B to invade a variety of cell types, while In1B interacts with the hepatocyte growth factor receptor, Met, on mammalian cells. This interaction is critical for infection. The examples provided herein demonstrate the basis for the invention by showing that rapidly after contact, In1B induces the mono-ubiquitination and endocytosis of Met and its subsequent degradation in a proteasome-independent manner. Moreover, overexpression or down-regulation of Cbl, the ubiquitin ligase that ubiquitinates Met, respectively increases or decreases bacterial invasion in epithelial cells, and RNAi-mediated knock down of major components of the endocytic machinery inhibits bacterial entry. Thus, the invention relies on the discovery that a bacterium induces and requires the ubiquitin-dependent endocytosis of its receptor to invade host mammalian cells. Furthermore, the invention is based on the knowledge that the In1B-induced degradation of Met is an extremely rapid process, being observed one minute after the addition of In1B, in contrast to activation with HGF, which causes a similar rate of Met degradation after two to five hours.
Based on this understanding, the invention provides In1B or In1B fragments or peptides, preferably those that interfere with the interaction between In1B and Met, as new cancer treatments that can achieve rapid down-regulation and subsequent degradation of Met. In addition, the invention provides screening tests to determine which compounds and In1B variants modulate the interaction between Met and In1B and/or interfere with Met signalling.
The invention is based on the discovery that a bacterium can trigger and exploit the monoubiquitin-dependent endocytosis machinery, to invade non-phagocytic mammalian cells. First, following In1B-dependent activation, Met becomes mono-ubiquitinated, and as with other RTKs, mono-ubiquitination of Met triggers its endocytosis. In addition, Listeria invasion depends on the endocytic machinery and Cbl, the ubiquitin ligase that ubiquitinates Met and other RTKs (5, 13-15), is needed for this invasion process. Overexpression of Cbl enhances, up to eight times, bacterial entry and down-regulation of Cbl, while dominant negative proteins or siRNA strongly inhibit bacterial entry, just as over expression or down-regulation of Cbl respectively increases or decreases endocytosis of RTKs (13, 14). The role of the ubiquitin is also highlighted by the ability of ubiquitin, or a ubiquitin that can only be mono-ubiquitinated (17), to increase bacterial entry by three-fold.
RNAi knock-down expression of different proteins involved in ligand induced RTK endocytosis including clathrin, eps15, Gbr2, dynamin, CiN85, CD2AP, cortactin, also inhibits Listerial entry, emphasizing the requirement of the endocytic machinery in the internalization/endocytosis of Listeria. The Examples provided herein show that the clathrin adaptor complex AP-2 is not involved in endocytosis of Listeria. Indeed, previous studies have shown that EGFR endocytosis is also independent of AP-2 (44, 45). In contrast, another clathrin adaptor complex, AP-1 (43) seems to be implicated in the entry of Listeria. Furthermore, knocking-down expression by RNAi of the clathrin adaptors GGA3 and Hrs, which are also ubiquitin-binding proteins (43), strongly inhibits Listeria endocytosis. GGA3 and AP-1 are involved in the trafficking between Golgi apparatus and endosomes (43, 46) and Hrs appears to associate with late endosomes or multivesicular bodies (47, 48), suggesting that the Golgi apparatus, endosomes, or both could provide membrane to allow the endocytosis of Listeria.
In1B-induced degradation of Met is extremely rapid, observable as soon as one minute after addition of In1B. In contrast, two or more hours are needed to achieve a similar rate of Met degradation after activation with HGF (19-21), indicating that In1B- and HGF-induced degradation of Met are different phenomena. Interestingly, Met is specifically and rapidly resynthesized after degradation, indicating that there must exist a yet unknown mechanism that couples the endocytic machinery to specific activation of the gene encoding the degraded protein.
Endocytosis is a cellular mechanism used to internalize receptors and other cargo molecules from the plasma membrane by invagination and pinching-off of membrane bound vesicles (49). In addition to macromolecules, it is also well known that large particles, such as viruses are able to get into the cell by endocytosis (50, 51). Moreover, the time required to complete assembly of the endocytic complex is proportional to the size of cargo molecules (36). The endocytosis of large particles, such as viruses, takes more time than for smaller molecules, such as transferrin. Accordingly, Cbl co-localizes with bacteria, as it is observed around activated receptor when endocytosis is slowed down (52).
The fact that bacteria, which are ten to fifty times larger than viruses, harness the endocytic machinery to invade non-phagocytic mammalian cells indicates that the size of cargo particles able to enter cells by endocytosis can be much larger than previously thought. Such large particles will also require a force to get in. An attractive candidate to generate this force is actin polymerization. Actin polymerization is indeed critical for Listeria invasion (53). It has been well established that actin polymerization during In1B-mediated entry involves the Arp2/3 complex in conjunction with proteins of WASP family shortly after In1B-mediated activation of Met (53). Interestingly, actin and actin-binding proteins play a critical role in yeast endocytosis and recent studies have pointed to a major role of the actin cytoskeleton in endocytosis in mammalian cells (38, 41). CD2AP binds Cbl and cortactin, which are able to directly activate the arp2/3 complex (42). It is now well established that dynamin can bind both directly and indirectly to activators of the Arp2/3 complex that are important for clathrin mediated endocytosis, including cortactin and the complex between N-WASP and syndapin. In addition, eps15 can recruit intersectin 1, which is also able to activate actin polymerization via activation of the arp2/3 complex (41). The clathrin coat can have a pivotal role supporting the invagination, transducing the compressive force generated by actin polymerization into pinching movement leading to fission at the neck of the phagocytic cup.
Thus, exploitation of the endocytic machinery can be a general mechanism used by bacteria to infect mammalian cells, as well as contributing to the formation of tumors.
In an embodiment of the invention, a method for treating a carcinogenic tumor in a mammal is provided. The method comprises administering a composition comprising In1B or a fragment of In1B to a mammal suffering from cancer, achieving degradation of Met in less than one hour, and reducing at least one carcinogenic aspect of the tumor.
In further embodiments, the carcinogenic aspects of the tumor include, but are not limited to, tumor growth, proliferation of tumor cells, angiogenesis in the tumor, invasiveness of the tumor, or dedifferentiated tumor morphology. “Tumor growth” means the expansion of the size of a solid tumor. “Proliferation of tumor cells” means the ability of the tumor cells to divide. “Angiogenesis” means the ability of a solid tumor to become vascularized. “Invasiveness” means the ability of the tumor to infiltrate non-tumor tissues. “Dedifferentiated” means that a tumor has lost the characteristics of the cell type it had before it became a tumor.
In other embodiments, the tumor can be a tumor of liver, kidney, breast, intestine, or other organs or tissues.
In yet further embodiments, the mammal that is treated by the method of the invention can be, but is not limited to, a dog, cat, cow, sheep, pig, goat, horse, monkey, or preferably, a human.
In another embodiment, the invention provides an anticancer treatment comprising a pharmaceutical composition comprising In1B or a fragment of In1B. In these embodiments of the invention, In1B or fragments of In1B are administered to the mammal. Fragments of In1B include, but are not limited to, peptides or small proteins derived from the GW domain or LRR domain of In1B. In preferred embodiments, the fragments are those described in (57), which are shown to be able to interact with Met and activate it. The LRR domain includes, but is not limited to, amino acids 76-248 of In1B. The GW domains include, but are not limited to, amino acids 392-630 of In1B. These domains, or subfragments of them, such as amino acids 464-630 of the GW domains, are also alternative embodiments for use in the claimed methods.
The In1B or a fragment of In B of the invention can be formulated as a pharmaceutical composition and administered in a variety of dosage forms adapted to the chosen route of administration, including, but not limited to, orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
Thus, the In1B or a fragment of In1B may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. It may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, fructose, lactose or aspartame or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring, such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.
The active compound can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the In1B or a fragment of In1B can be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer it to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid.
Useful solid carriers include finely divided solids, such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants, such as fragrances and additional antimicrobial agents, can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Useful dosages of the In1B or a fragment of In1B can be determined by correlating their in vitro activity, and in vivo activity in animal models, such as murine or dog models. The therapeutically effective amount of compound necessarily varies with the subject and the tumor to be treated. For example, doses of 10 μg to 1 g, 25 to 500 μg, 50 to 250 μg, 75 to 150 μg, or 30 to 50 μg can be used. The desired dose can conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
In another embodiment of the invention, a method of increasing the degradation rate of Met is provided. In this embodiment, a cell that contains Met is exposed to a composition comprising In1B or a fragment of In1B and the degradation rate of Met is increased in the cell. By decreasing the degradation rate of Met, certain characteristics of carcinogenic cells grown in culture may be modified. For example, but not to the exclusion of other characteristics, the rate of proliferation or degree of invasiveness or dedifferentiation of such cells may be modified to achieve a particular set of circumstances under which to perform experiments.
In yet another embodiment, the invention provides a method of screening for compounds that modulate the interaction between Met and In1B, comprising (A) adding a compound to a cultured cell; (B) incubating the cell for different times; (C) analyzing the Met protein in the cell after the different times of incubation; (D) determining which compounds alter the rate of degradation of Met in the cell; and (E) selecting the compounds that alter the rate of degradation of Met as ones that modulate the interaction between In1B and Met.
By way of example, but without limiting any parameters of this embodiment, the following screening method is provided: Semi-confluent HeLa cells growing in 6-well culture dishes are washed and purified In1B (5 nm) and the test compound or a control are added directly to the medium, i.e. DMEM. After 1, 2, 5, 10, 15, 30, 45, and 60 minutes incubation at 37° C., the cells are resuspended directly in SDS-PAGE sample buffer and immediately boiled. The samples are then separated on an SDS-PAGE gel and blotted with anti-Met monoclonal antibodies, according to protocols known to those in the art. The rate of degradation of Met is compared between the samples obtained from incubation in the presence or absence of the test compound. Samples that indicate the rate of Met degradation has changed indicate that the interaction between In1B and Met was modulated.
In yet more embodiments of the invention, the compounds that are identified by the screening method are provided as therapeutic anti-cancer treatments.
In yet another embodiment, the invention relates to a screening test for In1B variants that can interfere with Met signalling, based on the ability of Met to cause the phosphorylation of various proteins after interacting with HGF. In one screening test, mammalian cells expressing Met are grown in multiwell culture dishes, for example, but not limited to ELISA culture dishes with 96 or 384 wells. Medium containing HGF and at least one In1B variant, or a fragment thereof, is added to the wells of the culture dish, while some wells receive only HGF, as controls. After contact is made between HGF and the Met in the cells, the amount of protein phosphorylation is detected and quantified. Wells in which the In1B variant reduces the amount of phosphorylation observed in the control well indicate that the In1B variant interferes with Met signalling.
In the screening tests of this embodiment, phosphorylation can be, but is not limited to, tyrosine phosphorylation. In the case of tyrosine phosphorylation, quantification can be achieved using commercially available anti-phosphotyrosine antibodies directed against phosphotyrosine residues. These antibodies can be obtained from several companies, including, but not limited to, Cell Signaling Technology, Inc. and Upstate Group, LLC.
In an alternative embodiment of the screening test, phosphorylation of specific proteins can be measured. These proteins include, but are not limited to, phospho-Met, phospho-ERK, phospho-STAT3, and phospho-46JNK. Antibodies to detect phosphorylation of these proteins are also commercially available, for example from PhosphoSolutions, Inc.
In both of these embodiments of a screening test, the ability of In1B variants to reduce either total protein phosphorylation or the phosphorylation of specific proteins is indicative of the ability of the In1B variants to interfere with Met signalling.
In addition, the screening tests provided in these embodiments can be supplemented with a confirmation test in which In1B variants are analyzed in an invasion assay. Briefly, mammalian cells are gown on a collagen matrix. HGF is added, inducing the cells to separate and invade the matrix. The ability of In1B variants to inhibit Met signalling is tested by adding In1B variants, individually, to the wells containing invasive cells. Invasion is then measured visually using a microscope, a process which can be automated. In1B variants that inhibit the ability of the cells to invade the collagen matrix are confirmed as ones that inhibit Met signalling.
Throughout this application, various publications are referenced in parentheses by number. Full citations for these references may be found at the end of the specification immediately preceding the sequence listings and the claims. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
The following examples are presented to demonstrate the methods of the present invention and to assist one of ordinary skill in using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure.
Cells, bacteria and growth conditions. The human cell line used was HeLa (ATCC number CCL-2) grown as recommended by ATCC. Typically, cells were serum starved for three hours before the addition of In1B. The bacterial strains used were Listeria monocytogenes BUG600 and BUG1461. They were grown in BHI medium, which in the case BUG1641 contained 5 μg/ml of erythromycin.
Antibodies and reagents. The antibodies used were: rabbit polyclonal antibodies (pAb) anti-Met (C-12; used for immunoprecipitations), CD2AP (H-290), Grb2 (C-23), eps15 (H-896) from Santa Cruz, pAb anti Hrs (kind gift from Prof. Harald Stenmark), mAb anti-Met (DL21; used for immunoblots) from Upstate, mAb anti-ubiquitin (P4D1; Cell Signalling), mAb anti ubiquitin (FK1; Affinity), mAb anti β-actin (AC15; Sigma), mAb anti Ha (Santa Cruz), mAbs anti Cbl, AP50, Clathrin Heavy Chain, and GGA3 from BD pharmingen, pAbs anti-CIN85, dynamin II, (Calbiochem), rabbit serum anti Listeria (54). Reagents used were: cycloheximide (Merck), clasto-lactacystin β-lactone (Affinity), Epoxomicin (Affinity), Bafilomycin A1 (Upstate), ubiquitin ladder (poly-ubiquitin chains; Biomol), sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dipropionate (EZ-link Sulfo-NHS-SS-biotin; Pierce), sodium 2-mercapto sulfonate (MESNA; Fluka), Iodoacetamide (Sigma), immobilized NeutrAvidin (Pierce).
Plasmids. Plasmids codifying for Ha-tagged Cbl, v-Cbl (pMT2SM-Ha-c-Cbl, pMT2Ha-v-Cbl) and pMT2SM-Ha are as in Taher, T. E. I., et al., c-Cbl Is Involved in Met Signaling in B Cells and Mediates Hepatocyte Growth Factor-Induced Receptor Ubiquitination, J. Immunol., vol. 169, pp. 3793-3800 (2002). Plasmids encoding for Ha-Ubiquitin and Ha-monoubiquitin (K29, 48, 63R) are as in Haglund, K., et al., Multiple Monoubiquitination of RTKs is Sufficient for Their Endocytosis and Degradation, Nature Cell Biol., vol. 5, pp. 461-466 (2003).
Assays. Immunoprecipitation, Re-immunoprecipitation, and immunoblots were performed as described before (31). In1B purification was performed as described (54).
Cell surface biotinylation and internalization assay were performed as described before (35). Briefly, the cells were washed in ice-cold PBS, and incubated with 25 mg/ml EZ-link Sulfo-NHS-SS-biotin (Pierce) 15 minutes in PBS pH 8.0, and followed by one wash with serum free media containing 0.1% BSA, followed by one wash in PBS containing Ca and Mg (PBS+CaMg; Gibco). The cells were then warmed to 37° C., incubated with In1B for two minutes, and then cooled down to 4° C. The cells were washed again with PBS+CaMg, and cell-surface debiotinylation was performed by adding 100 mM MESNA in 50 mM Tris-HCl pH 8.6, 100 mM NaCl, 1 mM EDTA and 0.2% BSA (3 times, 10 minutes incubation). The cells were washed with PBS+CaMg, and residual MESNA was inactivated by a ten minute incubation with 120 mM iodoacetamide in PBS. Finally, the cells were then solubilized and precipitated with neutravidin agarose.
Immunoflorescence and differential immunofluorescent labelling of Listeria as well as gentamicin survival assays were performed as described before (54).
RNAi assays used double stranded RNA against: Cbl 5′-GGG AAA AGA MG MU GUA U-3′ (SEQ ID NO:1), cortactin 5′-GGG AGA AUG UCU UUC MG A-3′ (SEQ ID NO:2), Clathrin heavy chain 5′-GGC CCA GGU GGU MU CAU U-3′ (SEQ ID NO:3), Grb2 5′-GGU UUU GM CGA AGA AUG U-3′ (SEQ ID NO:4), eps15 5′-GGU UGA UAC AGG CM UAC U-3′ (SEQ ID NO:5), CIN85 5′-GGA GAG GUU UGU UCC CUG A-3′ (SEQ ID NO:6), GGA3 5′-GGA GGA CUG GGA AUA CAU A-3′ (SEQ ID NO:7), CD2AP 5′-GGA AUG UGA AAA AGC UAC A-3′ (SEQ ID NO:8), AP-50 (AP-2μ chain) 5′-GGA AAA CAU CM GM CM U-3′ (SEQ ID NO:9), and control RNA, (Silencer Negative Control 1 siRNA) were purchased from Ambion. Transfections were performed used oligofectamine (Invitrogen) as recommended by the manufacturer. Cells were tested 72 hours after transfections.
It has been previously described that the ubiquitin ligase Cbl is recruited to the Met complex during the first minute after cell treatment with soluble In1B (31). Whether Listeria infection promotes ubiquitination of Met was studied. Epithelial HeLa cells were infected with Listeria monocytogenes (at a m.o.i. 25) or treated with purified In1B (5 nM) (
To assess whether the signal observed after In1B treatment corresponded to Met ubiquitination, a re-immunoprecipitation assay was performed. Anti-Met immunoprecipitates from cells samples treated (or not) with In1B were denatured (boiled in presence of 1% SDS), to dissociate Met complexes, and re-immunoprecipitated using anti-Met antibodies. The resulting samples were immunoblotted against ubiquitin (
In order to determine the minimal amount of In1B necessary to trigger Met ubiquitination, HeLa cells were treated with increasing amount of In1B for two minutes. Cell extracts were then immunoprecipitated using the anti-Met antibody and blotted with anti ubiquitin or anti Met antibodies (
Next, the kinetics of In1B-dependent ubiquitination of Met were tested. In1B (5 nM) was added during increasing time periods to HeLa cells and cell extracts were immunoprecipitated using anti-Met antibodies (
Ligand-induced ubiquitination of RTKs is preceded by phosphorylation of the receptor, which is then recognized and ubiquitinated by the ubiquitin ligase Cbl (33). As previously shown (27), In1B rapidly stimulates Met phosphorylation and the kinetics of ubiquitination of Met follows that of phosphorylation with a maximum at two minutes after stimulation (
To study In1B-induced degradation of Met in more detail, HeLa cells were treated during increasing periods with In1B (5 nM) and anti-Met immunoblots were performed directly on total cell extracts (
In order to determine whether de-novo synthesis of Met occurred after In1B dependent-degradation, HeLa cells were pre-treated with cycloheximide, which inhibits protein synthesis, for 90 minutes before exposure to In1B. As showed in
Together these data show that degradation of Met after In1B contact is extremely rapid and also that In1B promotes de-novo and specific re-synthesis of Met. When In1B is present, de-novo synthesized Met was in turn ubiquitinated and then degraded (
The rapid degradation of Met after In B contact is different from the HGF-dependent degradation, which has been reported to take two hours (19-21), indicating differences in the biochemistry of the process. In addition, HGF-dependent ubiquitination of Met remains unclear, as it has been reported to be both poly-ubiquitination (15,20) or mono-ubiquitination (21). In orderto establish the nature of In1B-dependent ubiquitination of Met, both a monoclonal antibody (mAb), named FK1, which binds poly-ubiquitin but is unable to recognize mono-ubiquitinated proteins (17), and P4D1, a monoclonal antibody that recognizes both mono- and poly-ubiquitinated proteins, were used. Samples from HeLa cells treated with In1B (5 nM) were immunoprecipitated using an anti-Met antibody, and then Met-immunoprecipitates were analyzed with an anti Met mAb (
Cbl, the ubiquitin ligase that binds to Met and other RTKs and triggers their ubiquitination and further endocytosis after receptor-ligand contact (5, 15, 34), is recruited to the Met complex shortly after In1B addition to cells (31). These data indicated that the role of Cbl in the In1B-dependent ubiquitination and further degradation of Met should be analyzed further.
Short interfering RNA (siRNA) dramatically diminished Cbl expression, while expression of other cellular proteins, e.g. actin, was not affected (
Mono-ubiquitination is the signal that induces endocytosis of RTKs after ligand binding and usually drives cargo proteins to lysosomal degradation (6, 17, 34). To study whether In1B-dependent degradation of Met is a proteasomal or a lysosomal event, HeLa cells were treated with clasto-p-lactacystin lactone or epoximycin, two inhibitors of proteasome degradation, and ammonium chloride or bafilomycin A1, two inhibitors of lysosome degradation, one hour before the addition of In1B. Cellular extracts were then immunoblotted using anti Met antibodies (
To study whether In1B does induce endocytosis of Met, a biotin-based assay for internalization of cell surface proteins was used (35). Cell surface proteins were labelled with a biotin derivative that contains a disulphide bond and therefore can be cleaved by reducing agents. After biotin-labelling, cells were treated with In1B and allowed to internalize biotinylated Met. Cells were then treated with MESNA, a reducing agent unable to penetrate into the cell. After cell lysis, biotinylated, i.e. endocytosed proteins, were recovered by precipitation with neutravidin agarose and immunoblotted against Met. As shown in
As endocytosis of large particles, i.e. viruses, has been described to be slower than that of macromolecules (36), whether Listeria-induced endocytosis and degradation of Met differs from that following addition of soluble In1B was investigated. First the kinetics of Met degradation after Listeria addition to HeLa cells were followed. As showed in
Since In1B induces the ubiquitin-dependent endocytosis of Met, the localization of Met with entering bacteria suggests a direct coupling of Met endocytosis and bacterial entry.
To test this hypothesis, the role of Cbl in bacterial entry was further analyzed. HeLa cells that expressed an Ha-tagged version of Cbl (15) were infected with a superinvasive strain of Listeria (BUG 1641), which overexpress In1B covalently attached to its surface, thus improving the normal signals. The recruitment of Cbl by immunofluorescence was analyzed. These experiments clearly showed (
Furthermore, the relevance of ubiquitination in bacterial endocytosis was monitored by differential immunofluorescence labelling assay, on cells overexpressing ubiquitin and a ubiquitin mutant L 9, 48, 63 R only able to form monoubiquitin (17). As showed in
The first step in ligand-dependent endocytosis of RTKs after ligand contact is the Cbl dependent ubiquitination of the receptor, followed by recruitment of proteins belonging to the endocytic machinery and formation of a clathrin coated vesicle that is finally endocytosed (6, 10). Besides clathrin and clathrin-adaptors (38), other proteins involved in the ligand-induced endocytosis of Met and other RTKs. These include eps15, a ubiquitin binding protein that may be involved in the recruitment of clathrin to the pits (38), Grb2 an adaptor protein that can direct Cbl to activated receptors and is involved in the cargo entry into the clathrin coated pit (39, 40), dynamin a GTPase that directs the pinching off of the CCV from the membrane (41), CIN85 a Cbl binding protein that, through its interactions with endophilin, can induce membrane curvature (13, 14), CD2AP, a CIN85 related protein that also binds Cbl and cortactin, connecting the Cbl mediated endocytosis to the actin polymerization, as cortactin can activate the arp2/3 complex (42).
The role of these different components of the endocytic machinery in Listeria entry was examined. RNAi was used to inhibit expression (
Together, these data revealed that the bacterial protein In1B triggers the Cbl-dependent mono-ubiquitination of Met, coupling Met endocytosis, and Listeria entry into epithelial cells (see
Shortly after InIB treatment, Met is very rapidly degraded. Thus, whether InIB-induced degradation of Met is maintained for longer periods of time was tested. In fact, InIB-induced degradation of Met is maintained in time.
This result is significant because it shows that InIB degrades Met in the presence of HGF, which mimics the real environment in the body where InIB must act in the presence of HGF. In addition, the result shows that the degradation of Met is maintained during the time, impeding the HGF-Met contact for longer periods of time. Thus, InIB has very specific actions against Met and can be used in anticancer therapies.
Whether any differences in the phosphorylation state of specific proteins that are known to be phosphorylated as a consequence of HGF/Met interactions, for example, but not limited to, AKT and ERK, is tested.
In addition, whether InIB is able to inhibit the HGF/Met-dependent proliferation, movement and scattering is tested. Similarly, whether InIB inhibits in vitro the HGF/Met-induced invasion abilities of different cells lines is demonstrated.
In addition, in vivo experiments are performed that include the induction of HGF/Met dependent tumors in mice and the addition of InIB to reduce the size of the tumors and rescue the mice from death. Furthermore, models of HGF/Met dependent metastasis in mice are made and tested to determine if the addition of InIB reduces the number of the metastasis.
The entire disclosures of each of the following publications are relied upon and incorporated by reference herein:
The application claims the benefit of priority of U.S. Provisional Application No. 60/659,887 (attorney docket number 03495-6107), filed Mar. 10, 2005, which is incorporated herein by reference.
Number | Date | Country | |
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60659887 | Mar 2005 | US |