Patent Application
20140364402
This invention is directed to, inter alia, methods that are useful, for example, for identifying agents that selectively decrease the number of cancer stem cells in a population of cells.
This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file “SeqListing0311192.txt”, file size of 1.12 KB, created on Mar. 21, 2010. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(5).
Cancer is a term used to define a group of diseases characterized by unregulated cell proliferation, aberrant differentiation, and defective apoptosis. These neoplastic diseases may all have in common an initial transforming event that is manifold in nature (e.g., viral infection, chemical carcinogens, etc.) and impacts a unique tissue cell, the so-called “adult stem cell.” It is believed that these transforming events generate a “tumor/cancer stem cell (CSC)” that is responsible for tumor initiation and the hierarchical organization of cancer. However, such cell has not yet been definitively identified nor subsequently characterized.
Tumors consist of heterogeneous populations of cells that differ in growth capacities, morphology and marker expression. The cancer stem cell hypothesis posits that in addition to CSCs, tumors comprise a small population of transit amplifying clonogens, and large sets of differentiated malignant cells (Reya et al., 2008; Jordan et al., 2006; Dalerba et al., 2007; Vermeulen et al., 2008). Cancer stem cells, like their normal stem cell counterparts, should be undifferentiated and display asymmetrical cell division, resulting in self-renewal and production of differentiated clonogens, thus contributing to tumor heterogeneity. In addition, CSCs have been postulated to be refractory to standard chemotherapy treatments, though such treatments frequently result in eradication of the fastest dividing cells, resulting in tumor response, but not in CSC depletion (Gupta et al., 2009; Sharma et al., 2010; Bao et al., 2006; Trumpp et al., 2008; Dean et al., 2005; Costello et al., 2000; Guzman et al., 2002). This model explains why standard human cancer chemotherapy frequently results in initial tumor shrinkage, though most cancers eventually recur due to regeneration by surviving CSCs. Thus from a clinical point of view, the identification and molecular characterization of CSCs has fundamental implications for cancer diagnosis, prognosis and novel therapeutic approaches, including targeted treatments.
In solid tumors, CSCs have been previously claimed to be identified by cell surface immunophenotyping and tumor initiating capacity. These putative CSC subpopulations were shown to display a CD44+/CD24−/low and α6-integrin phenotype in breast cancer (Al-Hajj et al., 2003; Cariati et al., 2008; Fillmore et al., 2008; Ponti et al., 2005), a CD133+/Nestin+ phenotype in brain tumors (Singh et al., 2004), and a CD133+ phenotype in colon cancer (Ricci-Vitiani et al., 2007; O'Brien et al., 2007), among others. In prostate cancer, a similar subpopulation of putative CSCs that expresses high levels of CD44 has also been described (Collins et al., 2005; Patrawala et al., 2006; Li et al., 2008; Patrawala et al., 2007). Perhaps the most challenging issue facing the field is that the described CSC subpopulations do not always fulfill the classical properties that define “stemness”, mainly the ability to generate differentiated progeny by asymmetrical cell division. Furthermore, the existence of the above-mentioned surface phenotype subpopulations vary dramatically in tumor tissue samples from patients with the same histopathological diagnosis. In some cases, these subpopulations are relatively rare, whereas in others they constitute a large fraction of tumor mass (Quintana et al., 2008; Rosen et al., 2009; Chen et al., 2010; Shmelkov et al., 2008). These observations highlight the complexity of a consistent identification of CSCs.
Lack of consistency in identifying CSCs by their characteristics such as stemness has made a reliable search for agents targeting CSCs untenable. In particular, identification of agents that may modulate differentiation or other processes affecting the percentage of CSCs in a population of cells has not been feasible.
One embodiment of the present invention is a method for identifying an agent that selectively decreases the number of cancer stem cells (CSCs). This method comprises:
a. contacting a CSC from a population of cells with a candidate agent; and
b. determining whether the candidate agent reduces the survival or growth of the CSC or increases differentiation of the CSC relative to a CSC that has not been contacted with the candidate agent.
Another embodiment of the invention is a high throughput screening method for identifying agents that selectively decrease the percentage of cancer stem cells (CSCs) in a population of cells. This screening method comprises:
a. contacting a CSC from a population of cells with a candidate agent;
b. determining whether the candidate agent reduces the survival or growth or increases the differentiation of the CSC relative to a CSC that has not been contacted with the candidate agent by:
Another embodiment of the invention is a high throughput screening method for identifying agents that selectively decrease the percentage of cancer stem cells (CSCs) in a population of cells, wherein the CSCs are refractory to standard chemotherapy. This screening method comprises:
a. contacting a CSC from a population of cells with a candidate agent, wherein the CSC has the following properties: HLA I− and HLA II−;
b. determining whether the candidate agent reduces the survival or growth or increases differentiation of the CSC relative to a CSC that has not been contacted with the candidate agent by:
A further embodiment of the present invention is an agent for selectively killing CSCs identified by any methods of the present invention.
An additional embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier and an agent of the present invention.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
A CSC that is refractory to standard chemotherapy has been discovered. Although chemotherapy eradicates certain tumor cell subpopulations including dividing cells, it does not deplete the identified CSC population. This provides an explanation as to why standard chemotherapy frequently results in initial tumour shrinkage, yet most cancers eventually recur because of regeneration by surviving CSCs. Identifying an agent that reduces or eradicates the number of CSCs as defined herein would advance the field of cancer therapy.
A method in accordance with the present invention has been developed to identify an agent that selectively decreases the number of cancer stem cells (“CSCs”) having the phenotypical characteristics and functional chemoresistance of the CSCs that are identified herein. The in vitro screens of the present invention provide an assay for testing candidate agents to identify those that may reduce the survival or growth of the CSC or increase differentiation of the CSC. Implementation of the assays permits testing of a wide variety of candidate agents and facilitates the identification of agents with a putative CSC inhibitory effect.
Accordingly, one embodiment of the present invention is a method for identifying an agent that selectively decreases the number of cancer stem cells (CSCs). This method comprises:
a. contacting a CSC from a population of cells with a candidate agent; and
b. determining whether the candidate agent reduces the survival or growth of the CSC or increases differentiation of the CSC relative to a CSC that has not been contacted with the candidate agent.
A cancer stem cell (“CSC”) is a cancer cell that is HLA−. Such cells are further defined by being at least one of CD24−, CD133−, Notch+, Gli1+, Gli2+, glial fibrillary acidic protein− (GFAP−), Neurofil−, and cytokeratin−. Moreover, combinations of any or all of the foregoing are also contemplated. Such CSCs are further defined by having at least one of the following additional properties: capability to self-renew, undergo asymmetrical cell division, have tumorigenic capacity, have metastatic potential, have multi-differentiation properties, are sensitive to Notch and Hedgehog inhibitors, and have broad chemoresistance. Moreover, combinations of any or all of the foregoing are also contemplated. Further properties of CSCs are listed in, e.g., Table 1. The combination of any of the identified characteristics in Table 1 together with HLA− may be sufficient to identify a CSC cell.
Table 1 summarizes a number of biomarkers that further define the cancer stem cell phenotype. This phenotype may include expression of developmental pathways and biomarkers of “embryonic stem cells” and “tissue/adult stem cells,” such as certain homeobox regulatory factors (e.g., Sox2, Sox4), stem cell markers (e.g., ScaI, Gata2, Gata3, Nestin), transcription factors (e.g., Notch-2, GliI, Gli2, nuclear beta-catenin), asymmetrical cell division (e.g., RMND5A), and membrane transporters associated with multidrug resistance (e.g., MDRI/P-glycoprotein, MRPI, MRP2, MRP3, ABC3). Of major relevance, these CSCs have in common the lack of expression of histocompatibility proteins, including all HLA class I and HLA class II molecules, which contributes to the evasion of host immune-surveillance, thus being critical for the metastatic spread.
As used herein, “HLA I−” means having no major histocompatibility complex (MHC) class I molecules. The MHC is a set of molecules displayed on cell surfaces that are responsible for lymphocyte recognition and antigen presentation. The MHC class I molecules present antigen to cytotoxic T-cells. MHC I molecules are found on almost all types of body cells.
As used herein, “HLA II−” means having no major histocompatibility complex (MHC) class II molecules. The WIC class II molecules present antigen to helper T-cells. MHC II molecules are only found on macrophages, dendritic cells and B cells. “HLA−” preferably means HLA I− and HLA II−.
Identifying the HLA− cells from a cancer cell population may be accomplished using any conventional or known technique that identifies cells based on, e.g., expression, or non-expression, of particular cell surface markers and maintains the viability of the cell. Preferably, the identification is carried out by a Fluorescence-activated cell sorting (FACS) analysis as disclosed in more detail in the Examples.
As used herein, “capability to self-renew” means, e.g., that a CSC has the ability to go through numerous cycles of cell division while maintaining its undifferentiated state.
As used herein, “asymmetrical cell division” means, e.g., capable of having a cell division that leads to two cells with different properties, such as for example one cell which maintains the CSC phenotype while the other is programmed to, e.g., differentiate.
As used herein, “tumorigenic capacity” means, e.g., a cell, such as a CSC, that has the ability to generate a tumor when transplanted into a host animal.
As used herein, “metastatic potential” means, e.g., the ability of a cell, such as a CSC, to move to a secondary location in a body, i.e., metastasize, and generate a tumor.
As used herein, “multidifferentiation properties” means, e.g., the ability of a cell to differentiate into more than one cell type.
As used herein, “sensitivity to Notch and hedgehog inhibitors” means a cell, such as a CSC, which is modulated by inhibitors of the Notch and hedgehog pathways. Such inhibitors are known in the art. See, e.g., K. Garber, JNCI, 99(17):1284-1285 (2007) (Notch inhibitors) and Martinson et al., U.S. Pat. No. 7,695,965 (hedgehog inhibitors).
As used herein, “broad chemoresistance” means a cell, such as a CSC that is resistant to a range of chemical agents, such as, e.g., agents used to treat cancers. As used herein, an agent that is used to treat cancer is to be broadly interpreted and may include any known chemotherapy compound, composition or combination thereof. For example, the agent may be a DNA damaging drug and/or an anti-mitotic agent. Further non-limiting examples of the agent include: microtubulin inhibitors, topoisomerase inhibitors, vinblastine, vincristine, vinorelbine, paclitaxel, mitoxantrone, cisplatin, docetaxel, colchicines analogs, harringtonine, homoharringtonine, camptothecine, camptothecine analogs, podophyllotoxin, and combinations thereof.
As used herein, “a population of cells” means a mixture of different cells. For example, the CSCs do not have to be isolated from the rest of the cell types, such as, e.g., a population of cancer cells.
As used herein, “a population of cancer cells” or “a population of cancer/tumor cells” is to be broadly construed to include cancer/tumor cell lines, for example those which are readily available, through, e.g., the ATCC, and may be immortal (e.g., may be propagated indefinitely in vitro) and samples of, e.g., non-immortal cancerous material (“non-immortal samples”) obtained from a subject, such as a human, veterinary animal, or research animal such as a mouse, rat, etc. The cancerous material may be cells and/or tissue and/or fluid obtained, for example from a biopsy or other surgical procedure, from, e.g., a solid tumor, a blood-based tumor, or a nervous system tumor. Non-limiting examples of the source of the cancerous material also include blood, plasma, urine, cerebrospinal fluid, ascites fluid, tumor ascites, and combinations thereof.
Thus, a population of cancer cells may be obtained by harvesting cancer cells from a cell culture using well known techniques, including those disclosed in the Examples below. In addition, a population of cancer cells may be obtained through the acquisition of a tumor sample from a cancer patient. The population of cancer cells can be primary cancer cells or metastatic cancer cells. The cancer cells can be derived from a variety of tissue sources, e.g., prostate cancer cells, bladder cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, melanoma, or sarcoma. Further non-limiting examples of other such cancers that are within the scope of the present invention include leukemia, lymphoma, and glioma.
The population of cells in accordance with the invention may also be a mammalian CSC line that is enriched for cells that are HLA I− and HLA II−. “Mammalian” refers preferably to human, mouse, or other research mammal, such as e.g., a rat. As used herein, “enriched” means a cancer cell line that has increased numbers of CSCs relative to a control cell line that has not been treated, with e.g., an agent used to treat cancer. The increased numbers of CSCs may be transient, e.g., some of the CSC may differentiate, or may be of a longer duration. Moreover, such a cell line may or may not be pure, e.g., substantially free of HLA+ cells.
As noted above, CSCs may have at least one of the following properties: CD24−, CD133−, Notch+, Gli1+, Gli2+, GFAP−, Neurofil−, and cytokeratin−. Moreover, combinations of any or all of the foregoing are also contemplated. The CSCs may further have at least one of the following properties: capability to self-renew, undergo asymmetrical cell division, have tumorigenic capacity, have metastatic potential, have multi-differentiation properties, sensitive to Notch and Hedgehog inhibitors, and have broad chemoresistance. Moreover, combinations of any or all of the foregoing are also contemplated.
In one aspect of this embodiment, the CSCs may be refractory to standard chemotherapy. As used herein, “refractory” means resistant. For example, a sample of cancer/tumor cells may be derived from a sample of cancerous material of a subject that is resistant to chemotherapy, or the CSCs may be obtained from a cancer cell line that is resistant to chemotherapy. Methods for generating such resistant cancer cell lines are known in the art and disclosed in further detail in the Examples.
In another aspect of this embodiment, a candidate agent is selected from the group consisting of a chemical, a biologic, and combinations thereof. As used herein, a “biologic” means a substance which is derived from or produced by a living organism or synthesized to mimic an in vivo-derived agent or a derivative or product thereof. A biologic may be, for example, a nucleic acid, a polypeptide, or a polysaccharide. A “chemical” means a substance that has a definite chemical composition and characteristic properties and that is not a biologic. Non-limiting examples of chemicals include small organic compounds and small inorganic compounds. Non-limiting examples of candidate agents include Hedgehog and Notch inhibitors as disclosed in the Examples herein.
In an additional aspect of this embodiment, the determining step further comprises comparing the percentage of CSCs in a population of cells which is treated with the candidate agent to the percentage of CSCs in a population of cells which is not treated with the candidate agent, wherein a candidate agent that decreases the percentage of CSCs in the population of treated cells as compared to the percentage of CSCs in the untreated cells is an agent that selectively decreases the number of CSCs.
In another aspect of this embodiment, the determining step comprises carrying out a procedure selected from the group consisting of flow cytometry analysis, a cell viability assay, a cell differentiation assay, a cell division assay, and combinations thereof.
As set forth above, flow cytometry, such as FACS, may be used to separate and/or identify CSCs from non-CSC. It may also be used to assess cell viability, differentiation, or division.
As used herein, a “cell viability” assay is an assay that identifies and/or separates live cells from dead cells. A non-limiting example of a cell viability assay is disclosed in the Examples.
As used herein, a “cell differentiation” assay is an assay that identifies and/or separates differentiated cells or non-differentiated cells based on, for example, cell differentiation markers, such as, e.g., cytokeratin expression, as disclosed in the Examples.
As used herein, a “cell division” assay is an assay that identifies and and/or separates dividing cells from the non-dividing cells, for example, by performing a cell-cycle analysis, as disclosed in the Examples.
In another aspect of the embodiment, the population of cells comprises CSCs and adult stem cells, and the agent decreases the number of CSCs in the population of cells without substantially decreasing the number of adult stem cells. As used herein, “adult stem cells” means undifferentiated cells, found throughout the body after embryonic development, such as the body of an adult human, that multiply by cell division to replenish dying cells and regenerate damaged tissues. The adult stem cells serve as a repair system for the body. Thus, an agent that does not substantially decrease the number of adult stem cells will not substantially affect the body's normal repair system.
The methods of the present invention may be carried out using a high throughput screen. A high throughput screen is preferred to maxize the capacity to screen for potential compounds or biologics that reduce the number of CSCs.
The phrase “high throughput screen” or a “high throughput screening method” as used herein defines a process in which large numbers of agents, e.g., compounds, are tested rapidly and in parallel for biological activity against target molecules or target cells, such as, e.g., a CSC. In certain embodiments, “large numbers of agents, e.g., compounds” may be, for example, more than 100 or more than 300 or more than 500 or more than 1,000 compounds. Preferably, the process is an automated process.
In another aspect of this embodiment, the determining step may further comprise step i and at least one of steps ii, iii, and iv, as follows:
i. comparing the percentage of CSCs in a population of cells, which is treated with the candidate agent to the percentage of CSCs in a population of cells, which is not treated with the candidate agent;
ii. determining the percentage of viable cells in the treated and untreated population of cells;
iii. determining the percentage of non-CSC cells in the treated and untreated population of cells; and
iv. determining the percentage of dividing cells in the treated and untreated population of cells;
wherein a candidate agent that (1) decreases the percentage of CSCs and decreases cell viability; (2) decreases the percentage of CSCs without a decrease in cell viability; (3) decreases the percentage of CSCs and increases the percentage of non-CSCs; (4) decreases the percentage of CSCs and decreases cell division; or (5) decreases the percentage of CSCs without a decrease in cell division is an agent that selectively decreases the number of CSCs.
Preferably, the determining step comprises carrying out a procedure selected from the group consisting of flow cytometry analysis, a cell viability assay, a cell differentiation assay, a cell division assay, and combinations thereof.
Another embodiment of the present invention is a high throughput screening method for identifying agents that selectively decrease the percentage of cancer stem cells (CSCs) in a population of cells. This screening method comprises:
a. contacting a CSC from a population of cells with a candidate agent;
b. determining whether the candidate agent reduces the survival or growth or increases the differentiation of the CSC relative to a CSC that has not been contacted with the candidate agent by:
Step (i) of the present embodiment is preferably carried out by flow cytometry analysis.
Step (ii) of the present embodiment is preferably carried out by a cell viability assay.
Step (iii) of the present embodiment is preferably carried out by a cell differentiation assay.
Step (iv) of the present embodiment is preferably carried out by a cell division assay.
In another aspect of this embodiment, the candidate agent is selected from the group consisting of a chemical, a biologic, and combinations thereof.
In an additional aspect of this embodiment, the CSCs are refractory to standard chemotherapy.
A further embodiment of the invention is a high throughput screening method for identifying agents that selectively decrease the percentage of cancer stem cells (CSCs) in a population of cells, wherein the CSCs are refractory to standard chemotherapy. This method comprises:
a. contacting a CSC from a population of cells with a candidate agent, wherein the CSC has the following properties: HLA I− and HLA II−;
b. determining whether the candidate agent reduces the survival or growth or increases differentiation of the CSC relative to a CSC that has not been contacted with the candidate agent by:
In one aspect of this embodiment, the candidate agent is selected from the group consisting of a chemical, a biologic, and combinations thereof.
A further embodiment of the present invention is an agent for selectively killing CSCs identified by the methods disclosed herein.
Another embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the agent for selectively killing CSCs identified above or a pharmaceutically acceptable salt thereof.
Pharmaceutically acceptable salts as used herein are salts of the agent for selectively killing CSCs with physiologically compatible mineral acids, such as hydrochloric acid, sulphuric acid, sulphurous acid or phosphoric acid; or with organic acids, such as methanesulphonic acid, p-toluenesulphonic acid, acetic acid, lactic acid, trifluoroacetic acid, citric acid, fumaric acid, maleic acid, tartaric acid, succinic acid or salicylic acid. The term “pharmaceutically acceptable salts” refers to such salts. Agents in which an acidic group is present can further form salts with bases. Examples of such salts are alkaline, earth-alkaline and ammonium salts such as e.g., Na—, K—, Ca— and trimethylammoniumsalt. The term “pharmaceutically acceptable salts” also refers to such salts.
A pharmaceutical composition of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a pharmaceutical composition of the present invention may be administered in conjunction with other treatments. A pharmaceutical composition of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.
The pharmaceutical compositions of the invention comprise one or more active ingredients, e.g. one or more agents identified by the methods of the present invention, in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).
Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
The pharmaceutical compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in such pharmaceutical compositions. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.
Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.
Pharmaceutical compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Pharmaceutical compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.
Pharmaceutical compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Nucleic Acid
“Nucleic acid” or “oligonucleotide” or “polynucleotide” used herein mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be synthesized as a single stranded molecule or expressed in a cell (in vitro or in vivo) using a synthetic gene. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
The nucleic acid may also be a RNA such as a mRNA, tRNA, shRNA, siRNA, Piwi-interacting RNA, pri-miRNA, pre-miRNA, miRNA, or anti-miRNA, as described, e.g., in U.S. patent application Ser. Nos. 11/429,720, 11/384,049, 11/418,870, and 11/429,720 and Published International Application Nos. WO 2005/116250 and WO 2006/126040.
siRNA gene-targeting may be carried out by transient siRNA transfer into cells, achieved by such classic methods as lipid-mediated transfection (such as encapsulation in liposome, complexing with cationic lipids, cholesterol, and/or condensing polymers, electroporation, or microinjection). siRNA gene-targeting may also be carried out by administration of siRNA conjugated with antibodies or siRNA complexed with a fusion protein comprising a cell-penetrating peptide conjugated to a double-stranded (ds) RNA-binding domain (DRBD) that binds to the siRNA (see, e.g., U.S. Patent Application Publication No. 2009/0093026).
The nucleic acid may also be an aptamer, an intramer, or a spiegelmer. The term “aptamer” refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by EXponential Enrichment), disclosed in U.S. Pat. No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2′-OH group of a ribonucleotide may be replaced by 2′-F or 2′-NH2), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term “intramer” refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those disclosed in U.S. Pat. Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within the definition of nucleic acid. The modified nucleotide analog may be located for example at the 5′-end and/or the 3′-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2′-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as disclosed in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Application Publication No. 20050107325. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as disclosed in U.S. Patent Application Publication No. 20020115080. Additional modified nucleotides and nucleic acids are disclosed in U.S. Patent Application Publication No. 20050182005. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
Peptide, Polypeptide, Protein
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein. In the present invention, these terms mean a linked sequence of amino acids, which may be natural, synthetic, or a modification, or combination of natural and synthetic. The term includes antibodies, antibody mimetics, domain antibodies, lipocalins, targeted proteases, and polypeptide mimetics. The term also includes vaccines containing a peptide or peptide fragment intended to raise antibodies against the peptide or peptide fragment.
“Antibody” as used herein includes an antibody of classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab′)2, Fd, and single chain antibodies, diabodies, bispecific antibodies, and bifunctional antibodies. The antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. The antibody may also be a chimeric antibody. The antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art. The antibody may be conjugated with a chemical moiety. The antibody may be a human or humanized antibody. These and other antibodies are disclosed in U.S. Published Patent Application No. 20070066447.
Other antibody-like molecules are also within the scope of the present invention. Such antibody-like molecules include, e.g., receptor traps (such as entanercept), antibody mimetics (such as adnectins, fibronectin based “addressable” therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.), domain antibodies (the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15; 64(8):2853-7)).
Suitable antibody mimetics generally can be used as surrogates for the antibodies and antibody fragments described herein. Such antibody mimetics may be associated with advantageous properties (e.g., they may be water soluble, resistant to proteolysis, and/or be nonimmunogenic). For example, peptides comprising a synthetic beta-loop structure that mimics the second complementarity-determining region (CDR) of monoclonal antibodies have been proposed and generated. See, e.g., Saragovi et al., Science. Aug. 16, 1991; 253(5021):792-5. Peptide antibody mimetics also have been generated by use of peptide mapping to determine “active” antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, so as to design a peptide mimic containing antigen contact residues from multiple CDRs. See, e.g., Cassett et al., Biochem Biophys Res Commun. Jul. 18, 2003; 307(1)198-205. Additional discussion of related principles, methods, eta, that may be applicable in the context of this invention are provided in, e.g., Fassina, Immunomethods. October 1994; 5(2):121-9.
As used herein, “peptide” includes targeted proteases, which are capable of, e.g., substrate-targeted inhibition of post-translational modification such as disclosed in, e.g., U.S. Patent Application Publication No. 20060275823.
In the present invention, “peptide” further includes anticalins. Anticalins can be screened for agents that decrease the number of cancer stem cells. Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In general, a polypeptide mimetic (“peptidomimetic”) is a molecule that mimics the biological activity of a polypeptide, but that is not peptidic in chemical nature. While, in certain embodiments, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids), the term peptidomimetic may include molecules that are not completely peptidic in character, such as pseudo-peptides, semi-peptides, and peptoids. Examples of some peptidomimetics by the broader definition (e.g., where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below. Whether completely or partially non-peptide in character, peptidomimetics according to this invention may provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in a polypeptide. As a result of this similar active-site geometry, the peptidomimetic may exhibit biological effects that are similar to the biological activity of a polypeptide.
There are several potential advantages for using a mimetic of a given polypeptide rather than the polypeptide itself. For example, polypeptides may exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action. Peptidomimetics are often small enough to be both orally active and to have a long duration of action. There are also problems associated with stability, storage and immunoreactivity for polypeptides that may be reduced with peptidomimetics.
Polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities. Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure, shape or reactivity. The development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling. These techniques aid in the development of novel compositions of higher potency and/or greater bioavailability and/or greater stability than the original polypeptide (Dean (1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98.
Polysaccharides
The term “polysaccharides” means polymeric carbohydrate structures, formed of repeating units (either mono- or di-saccharides) joined together by glycosidic bonds. The units of mono- or di-saccharides may be the same or different. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, and chitin.
Small Organic or Inorganic Molecules
The phrase “small organic” or “small inorganic” molecule includes any chemical or other moiety, other than polysaccharides, polypeptides, and nucleic acids, that can act to affect biological processes. Small molecules can include any number of therapeutic agents presently known and used, or can be synthesized in a library of such molecules for the purpose of screening for biological function(s). Small molecules are distinguished from macromolecules by size. The small molecules of this invention usually have a molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, most preferably less than about 500 Da.
As used herein, the term “organic compound” refers to any carbon-based compound other than macromolecules such as nucleic acids and polypeptides. In addition to carbon, organic compounds may contain calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements. An organic compound may be in an aromatic or aliphatic form. Non-limiting examples of organic compounds include acetones, alcohols, anilines, carbohydrates, mono-saccharides, di-saccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters, ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds, imidizoles, and phenols. An organic compound as used herein also includes nitrated organic compounds and halogenated (e.g., chlorinated) organic compounds. Collections of small molecules, and small molecules identified according to the invention are characterized by techniques such as accelerator mass spectrometry (AMS; see Turteltaub et al., Curr Pharm Des 2000 6:991-1007, Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research; and Enjalbal et al., Mass Spectrom Rev 2000 19:139-61, Mass spectrometry in combinatorial chemistry.)
Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics of the chemical entity.
The following examples are provided to further illustrate the methods and compositions of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
Docetaxel is an anti-mitotic agent currently used as standard therapy in patients with hormone refractory prostate cancer (Petrylak et al., 2004; Tannock et al., 2004. However, all patients ultimately experience disease progression, and no other treatment controls the disease in this context. Due to the clinical relevance of this resistance phenomenon, an in vitro model of Docetaxel resistance using the well established prostate cancer hormone-independent cell lines DU145 and 22RV1 was generated to characterize the molecular alterations responsible for such an event. (
DU145 and 22RV1 cells were exposed to increasing doses of Docetaxel, and the acquired chemoresistance was characterized and confirmed by cell viability, colony formation, annexin V, and poly-(ADP-ribose) polymerase (PARR) cleavage assays. (
Gene expression profiling, using oligonucleotide microarrays, was performed to compare sensitive (DU145 and 22RV1-parental cells) and acquired resistant (DU145-DR and 22RV1-DR) prostate cancer cells. Genes with at least 2 fold increase or decrease in transcript expression were selected for further analysis. This analysis disclosed 1245 and 990 deregulated genes in DU145-DR and 22RV1-DR cells, respectively, of which 247 genes overlapped (
laevis)
Genes that were found up- or down-regulated in Docetaxel resistant cells (DU145-DR and 22RV1-DR) when compared to their parental sensitive cells DU145 and 22RV1) are summarized in Table 2. Statistical analysis of the mean expression average difference of genes, which show ≧2 fold change based on a logarithmic normalization, was done using a t-test between matched sensitive and resistant cells. There was a decrease in the transcription levels of genes involved in epithelial differentiation, prostate specific markers and immune-surveillance markers. On the other hand, gene transcripts of developmental transcription factors were up-regulated.
Protein validation of these genes by western blot (
With respect to studies on differentiation, the expression of cytokeratins (CKs) as epithelial markers, as well as prostate-related biomarkers, including AR, PSA and PSMA, were assessed. CKs have been previously reported to be specific for human differentiated epithelial cells, playing a role in the maintenance of cellular integrity while also functioning in signal transduction and cellular differentiation processes (Brulet et al., 1980; Lu et al., 1980; Oshima et al., 1981; Tesar et al., 2007). Low molecular weight CKs (e.g., CK18, and CK19) are specifically expressed in luminal normal human prostate cells and prostate cancer, whereas high molecular weight CKs (e.g., CK5 and CK10) are identified in basal normal prostate cells and rarely observed in cancer cell populations (Ali et al., 2008). Docetaxel resistant cells showed a significant decrease in both gene transcription and protein expression of low molecular weight CKs. DU145-DR cells showed a 6.25 and 16.6 fold decrease in the protein expression of CKs 19 and 18 respectively. Similarly, 22RV1-DR showed a 14.3 and 6.7 fold decrease in such CKs when quantified and compared to the sensitive parental cells. Immunofluorescence staining of CK19 and CK18 confirmed their decreased expression in the Docetaxel resistant cells (
Regarding mechanisms of immune evasion, it was found that Docetaxel resistant cells showed deregulation of WIC class I molecules. It was observed that MHC class I antigens, which are critical for efficient antigen presentation to cytotoxic T lymphocytes and subsequent tumor cell lysis, were down-regulated at both gene transcription and protein level (
Regarding studies on stem cell phenotype, the identified deregulated expression of WNT/β-catenin, Notch and Hedgehog was characterized. These pathways have been implicated in self-renewal and differentiation of progenitor cells (Katoh et al., 2007; McDonald et al., 2006; van den Brink et al., 2004; Radtke et al., 2006; Leong et al., 2008; and Grigoryan et al., 2008). In the prostate, these signaling pathways play essential roles in developmental patterning, epithelial regeneration, and prostate cancer tumorigenesis (Wang et al., 2006, Karhadkar et al., 2004). Docetaxel resistant cells showed a significant decrease in both gene transcript and protein levels of the WNT inhibitor Dickkopf-I (DKKI), a well known inhibitor of the WNT/β-catenin signaling network (Fedi et al., 1999). This decrease in DKK1 expression was linked to an increase in the expression of de-phosphorylated (active) β-catenin, which is the major key effector of WNT signaling. immunofluorescence analyses demonstrated that parental Docetaxel sensitive cells displayed a membranous expression of β-catenin, associated to its function as an adhesion molecule, whereas Docetaxel resistant cells showed a pronounced nuclear localization of this protein (
Based on the fact that the Docetaxel resistant cells exhibited a phenotype consistent with stemness, whether these cells displayed a higher tumor initiating capacity than the parental cells was tested. Subcutaneous injection in NOD/SCID mice of 102 and 103 Docetaxel resistant cells (22RV1-DR and DU145-DR) gave rise to significantly more tumors than their parental sensitive cells (
Considering the stemness signature and the higher tumor initiating capacity of the generated Docetaxel resistant cells, it was investigated whether the chemoresistance phenomenon was due to a transition of sensitive to resistant cells with stemness characteristics, or alternatively if chemotherapy would select for pre-existing prostate cancer stem cells (
It was then studied whether the identified CK-negative/HLA class I-negative tumor cells could survive Docetaxel exposure, thus being responsible for the acquired chemoresistance phenomenon. For this purpose, DU145 parental cells were stably transfected with a plasmid containing the promoter of CK19 driving the expression of the green fluorescence protein (GFP). (
Unsorted DU145-CK19 promoter-GFP stable cells were seeded and exposed to Docetaxel (10 nM) and Live imaging was performed for periods of up to 48 hours. The resulting movies showed that Docetaxel exposure selected for cells with the GFP-negative phenotype which were able to divide and exit mitosis under therapy, whereas GFP-positive cells died after mitotic arrest (
It was also demonstrated that the subpopulation of tumor cells with a CK-negative/HLA class I-negative/GFP-negative phenotype had the unique stem cell property to differentiate through asymmetric cell division. When seeding unsorted DU145-CK19 promoter-GFP stable cells, it was observed that through such process GFP-negative cells give rise to daughter cells that enter the program of differentiation (become GFP-positive), while the GFP-negative cell retains its original identity (
Finally, the functional cancer stem cell property of tumor initiation in the identified subpopulations of cells was addressed. DU145-CK19 promoter-GFP stable cells were sorted by GFP/HLA class I expression and the obtained GFP sorted subpopulations of cells were injected subcutaneously into immunodeficient NOD/SCID mice and only the GFP-negative/HLA class I-negative cells exhibited tumor initiating capacity (
In order to further confirm the observed findings in which only the DU145 CK-negative/HLA class I-negative/GFP-negative phenotype cells showed tumor initiating capacity, parental cell lines (DU145 and 22RV1) were sorted based on the expression of surface marker HLA class I antigen and their tumor initiating capacity was tested. (
Thus, the functional cancer stem cell property of tumor initiation was intrinsic to the subpopulation of cells with a CK-negative/HLA class I-negative phenotype. Furthermore, in addition to being the only cells endowed with tumour initiating capacity, HLA class l-negative cells also displayed a statistically significant higher clonability capacity when compared to HLA class I-positive cells (
Taken together, in vitro and in viva results identify a cell population with a CK-negative/HLA class I-negative phenotype with tumor initiating capacity, which through asymmetrical cell division generates transit amplifying clonogens that overwhelmingly populate the evolving tumor with differentiated cells. Thus, confirming the discovery of a prostate cancer stem cell. Considering all of the results described above, including the stemness signature and the tumor initiating capacity of the newly generated Docetaxel resistant cells, it was hypothesized that chemotherapy induces an enrichment of prostate cancer stem cells (
In view of the above results, it was investigated whether the identified prostate cancer stem cell population was present in human prostate cancer tissue samples. Immunohistochemical studies of metastases (n=20) and matched primary (n=6) human prostate cancer tissues revealed a scattered subpopulation of CK-negative tumoral cells (
All human prostate cancer specimens analyzed contained scattered subpopulations of CK-negative (CK18 and CK19) tumor cells, accounting for 0.05% to 0.3% and 0.4% to 1.8% of all tumor cells in primary and metastatic lesions, respectively (
Given that the cancer stem cell population was identified in both primary and metastatic prostate cancer, a series of experiments were designed aimed at investigating the tumorigenic ability of such population of cells from fresh human tumor samples. To assess whether prostate cancer cells with such phenotype were responsible for tumor initiation, tumors from 48 patients who underwent radical prostatectomy for primary prostate cancer were obtained. (Table 4). Cells were isolated by flow cytometry based in the expression of the cell surface marker HLA class I (
Table 5 summarizes the tumour initiating capacity measured by tumour incidence (tumours/injections) and tumour latencies in weeks (mean±SD), when 10, 100 and 1000 HLA class I sorted and unsorted cells from primary prostate cancer tissues were injected. Four mice for each sorted cell population and cell dilution were injected twice in the upper flanks (HLA-negative) and lower flanks (HLA-positive). Unsorted cells from each tumour specimen were also injected.
After primary injection of 103 cells, it was observed that a significantly higher tumor initiating capacity was from the HLA-negative sorted cells (87.5±17.6%) when compared to the HLA-positive cells (12.5±17.6%), a fact that was linked to a lower tumor latency in the HLA-negative xenografts (14.2±4.5 versus 24.6±3.1 weeks). Moreover, further dilution into 102 and 103 cells revealed that only the HLA-negative cells were endowed with tumor initiating capacity. These observations were confirmed by secondary transplantation experiments in which injection of 102 and 103 sorted cells continued to demonstrate that tumor development was restricted to the HLA-negative subpopulation of cells (
Moreover, it appears that this is a universal phenomenon, because HLA-negative tumor cells were isolated from a variety of fresh human solid neoplasms, including colon, breast, lung and bladder carcinomas which are responsible for tumor initiation when serially injected into NOD/SCID mice (
Table 8 summarizes the tumour initiating capacity measured by tumour incidence (tumours/injections) and tumour latencies in weeks (mean±SD), when 100 HLA class I sorted cells from other primary tumour tissue types were injected. Four mice for each sorted cell population and cell dilution were injected twice in the upper flanks (HLA-negative) and lower flanks (HLA-positive).
After primary injection, tumor initiating capacity of 102 HLA-negative cells was significantly higher when compared to HLA-positive sorted cells. More importantly, following serial transplantation only the HLA-negative cells retained tumorigenic capacity, whereas HLA class I-positive cells did not. Moreover, secondary injections of HLA-positive cells sorted from xenografts generated from HLA-positive cells did not possess tumour initiating capacity. The generation of tumours from HLA class I-positive cells could occur because of possible contamination of HLA class I-negative cells during cell sorting, although it cannot be excluded that HLA class I-positive cells may have a low tumour initiating capacity that cannot be maintained after serial transplantation. Histological analysis of the developed xenograft tumors showed similar morphological characteristics than their corresponding human primary cancers (
Finally, the tumor initiating capacity of cells from fresh human tissue samples was not related to any of the analyzed clinico-pathological characteristics of the cancer patients, neither associated with the percentage of HLA-negative cells. Tumours with aggressive clinico-pathological characteristics (e.g., high grade, high tumour stage) or tumours with a high number/percentage of HLA-negative cells (e.g., 1.5%) did not possess a significantly higher tumour initiating capacity. Moreover, no association was observed between the percentage of HLA-negative cells and either tumour stage or tumour grade.
Based on the fact that the identified cancer stem cells exhibited Hedgehog and Notch pathways activation, testing was done as to whether inhibition of such pathways could impair cancer stem cell homeostasis. For this purpose, Cyclopamine, a plant derived hedgehog pathway antagonist that acts at the level of Smo (Taipale et al., 2000; Karhadkar et 2004; Chen et al., 2002), was utilized. Compound E is a highly active gamma-secretase inhibitor that blocks the proteolytic processing of Notch receptors (Seiffert et al., 2000). For the in vitro experiments, DBZ, a highly active gamma-secretase inhibitor with established in vivo activity, was used as a substitute for Compound E in the in vivo experiments outlined below (van Es et al., 2005).
Exposure of the CK-negative/HLA-negative cancer stem cell population from DU145 and 22RV1 to each individual inhibitor did not induce any major effect. However, when both agents were administered together a robust inhibition in cell cycle progression with an accumulation of cells in sub-G1 was observed, whereas this effect was minor in differentiated (CK-positive/HLA-positive) cells (
Finally, the tumor initiating inhibitory effects of these compounds was tested in xenografts derived from fresh human prostate cancer tissues 103 HLA-negative sorted cells from xenografts #5, #9 and #12 were injected into NOD/SCID mice and treated with the same schedules and combinations of Hedgehog and Notch inhibitors, as described above. As observed previously in the cell lines, only the combined treatment with Hedgehog and Notch inhibitors significantly (p<0.05) delayed tumor initiation (
Taken together, these results show that the inhibition of these developmental pathways targets the cancer stem cell population delaying tumor initiation.
Two major hypotheses regarding tumor initiation have been postulated. The “stochastic model” which predicts that every neoplastic cell can generate an entirely new tumor; and the “cancer stem cell model” which proposes that tumor cells exist in a hierarchical state, and that only a few stem cells possess tumor initiating potential. The identification and functional characterization of a human cancer stem cell is disclosed, which fulfills the following stemness criteria: 1) self-renewal and differentiation through asymmetrical cell division, 2) tumor initiating capacity, 3) a negative histocompatibility signature, and 4) a multidrug resistance phenotype.
An HLA-negative phenotype is also shared by embryonic stem cells. It has been reported that human pre-implantation embryos are HLA class I and class II negative (Desoye et al., 1988). This phenomenon precludes rejection based on expression of paternal antigens, until a blood-tissue barrier develops, in this situation being the placenta. In the context of cancer stem cells, such a histocompatibility negative phenotype has major clinical implications, since it explains host mutation permissiveness, as well as tumor spread and metastatogenic capabilities, since cancer stem cells would escape immune-surveillance.
Concerning tumor initiation capacity, it is disclosed that only the identified CK-negative/HLA-negative cancer stem cells generate tumors in viva, while the differentiated, CK-positive/HLA-positive progenies lack such property. However, it appears that these cancer stem cells exhibit genetic memory independent of certain stroma interactions, since subcutaneous injections confer the tissue-of-origin phenotype without the need for orthotopic implantation, a phenomenon that needs to be further investigated. Moreover, due to its homogenous phenotype, it was hypothesized that these cells are genetically stable, a property facilitated by their quiescent state and asymmetrical division. This genetic stability would allow for the identification of “driver” mutations, since molecular heterogeneity would be essentially a product of the tumor expanding and differentiated cancer cell populations, and probably not as critical for tumorigenesis. A new molecular classification of human tumors could probably be derived from analysis of such “driver” mutations in these cancer stem cell populations.
In sum, a newly defined prostate cancer stem cell in human cancer cell lines and tissue samples has been identified and characterized. Moreover, this population of cells was isolated using HLA class I surface marker, and its tumor initiating capacity was demonstrated. Further, it was observed that similar CSC populations are also present in human breast, colon, lung and bladder carcinomas, a fact that gives further universality to these findings. The discovery of this human cancer stem cell has important clinical implications in diagnostic and predictive laboratory assays, as well as for development of novel therapeutic strategies. In this context, treatment with Notch and Hedgehog inhibitors attenuates tumor formation in experimental animal models.
Docetaxel, Mitoxantrone, Doxorubicin, Cisplatin, Vinorelbine, Paclitaxel, Dexamethasone, Cyclopamine and Compound E were obtained from Sigma-Aldrich (St. Louis, Mo.). DBZ [(2S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,-d]azepin-7-yl)-propionamide] was obtained from Syncom (Groningen, The Netherlands).
Human hormone-independent prostate cancer cell lines, DU-145 and 22RV1, were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium (Gibco, Invitrogen Corp., Carlsbad, Calif.) supplemented with 10% FBS without antibiotics. Cells were grown at 37° C. in a humidified atmosphere with 5% CO2. DU145 and 22RV1 cells were selected in order to generate a prostate cancer Docetaxel resistance model. This selection was based on the fact that both cell lines are hormone-refractory, a condition treated with Docetaxel in the clinical setting, and that they also exhibit distinct hormone-refractory phenotypes. While 22RV1 cells are still dependent of androgen receptor signaling and express prostate specific markers (e.g., PSMA, androgen receptor), DU145 do not. Thus, the generation of acquired Docetaxel resistance in these two phenotypically distinct cell lines facilitated an approach to study the molecular processes involved in the acquisition of Docetaxel resistance. Docetaxel resistant clones, DU-145-DR and 22RV1-DR, were selected by culturing cells with Docetaxel in a dose-escalation manner. Initial culture was at 5 nM Docetaxel. After the sensitive clones were no longer present and the surviving DU-145 and 22RV1 cells repopulated the flask, the concentration of Docetaxel was increased to 10 nM and subsequently to 25 nM, 50 nM, 100 nM and 250 nM. 22RV1-DR cells were further exposed to 500 nM Docetaxel. After exposure to each increasing dose of Docetaxel, the remaining surviving cells were maintained in culture medium containing the last selection escalating dose of Docetaxel. The last drug selection concentration at which the cells were exposed was 250 nM for DU-145-DR and 500 nM for 22RV1-DR, in order to avoid reversibility of the acquired Docetaxel resistance phenotype. The process of acquired drug resistance took 9 months for DU-145-DR and 6.5 months for 22RV1-DR. In parallel, parental DU-145 and 22RV1 cells were exposed to DMSO (vehicle solution of Docetaxel) in the same dose-escalation manner.
Cells were exposed to increasing doses of Docetaxel, and the acquired chemoresistance was characterized and confirmed by cell viability, colony formation, annexin V, and poly-(ADP-ribose) polymerase (PARP) cleavage assays (
Cell viability was analyzed using the Cell titer 96 Aquos Non-Reactive Cell Proliferation Assay (MTS) kit (Promega Corp., Madison, Wis.). Cells were seeded at a density of 104 in 96-well culture dishes and 24 hours later medium was removed and replaced with new medium alone (control) or medium containing drugs. After 72 hours, color absorbance was measured on a microplate spectrophotometer (Molecular Dynamics) at 450 nm (test wavelength) and 620 nm (reference wavelength). The percentage of surviving cells was estimated by dividing the A 450 nm-A 620 nm of treated cells by the A 450 nm-A 620 nm of control cells. Clonogenic survival assays in response to drug treatment were performed by plating 103 cells in 35 mm culture dishes. After 24 hours, cells were left untreated (control) or treated with drugs. Next day, medium was changed and the cells kept growing in fresh medium without any drug or under continuous exposure to drugs. For these continuous exposure experiments, medium plus drugs were replaced every 3 days until clones of drug-resistant cells appeared. Cells were then fixed with 4% paraformaldehyde in PBS, stained with crystal violet solution and formed colonies were visually counted.
Cells (105) were left untreated (control) or treated with drugs for 72 hours. Adherent and detached cells were pooled, washed and labeled with annexin-V-FITC and propidium iodide using the annexin-V-FLUOS Staining Kit (Roche, Nutley, N.J.) according to manufacturer's instructions. Samples were acquired with a FACscan Flow Cytometer (BD Biosciences, San Jose, Calif.) and analyzed with CellQuest Pro software (BD Biosciences) to determine the percentage of cells displaying annexin V staining.
Cells were treated with drugs for 72 hours, harvested and fixed in 70% ethanol and stored at 4° C. Before analysis, cells were washed with PBS, centrifuged and incubated for 30 min at room temperature in a staining solution containing 0.1% Triton X, 0.2 mg/ml RNAse and 0.02 mg/ml propidium iodide. DNA content was acquired with a FACscan Flow Cytometer (BD Biosciences) and analyzed with CellQuest Pro software (BD Biosciences).
cDNA Microarray Analysis
22RV1, 22RV1-DR, DU-145 and DU-145-DR gene expression profiles were analyzed. Total RNA from each sample was isolated by Tryzol (Invitrogen) and purified by RNeasy mini kit and RNase-free DNase set (Qiagen Inc., Valencia, Calif.) according to the manufacturer's protocols. RNA quality of all samples was tested by RNA electrophoresis and RNA LabChip analysis (Agilent Technologies, Inc., Santa Clara, Calif.) to ensure RNA integrity. Samples were prepared for analysis with Affymetrix Human U133 Plus 2.0 arrays according to the manufacturer's instructions. Gene expression levels of samples were normalized and analyzed with Microarray Suite, MicroDB, and Data Mining tool software (Affymetrix, Santa Clara, Calif.). The absolute call (present, marginal, or absent) and average difference of 22.215 expressions in a sample, and the absolute call difference, fold change, average difference of gene expression between two or three samples were normalized and identified using this software package. Statistical analysis of the mean expression average difference of genes, which show ≧2-fold change based on a log normalization, was done using at test between Docetaxel sensitive and resistant samples. Genes that were not annotated or not easily classified were excluded from the functional clustering analysis.
Genes differentially expressed in the Docetaxel resistant cells compared to the parental sensitive cells generated a list of commonly deregulated transcripts. This list was assessed by the DAVID Bioinformatics Resources, a web-based statistical hypergeometric test applied for enrichment analysis of gene ontology (GO) categories, which are, biological process, molecular function, and cellular component. GO categories enriched on the highest hierarchical level (≧level 5) at statistical significance (p<0.01) were taken into consideration.
Whole cell extracts were prepared in sample buffer and analyzed by immunoblotting. Primary antibodies against poly (ADP-ribose) polymerase (PARP) (BD Pharmingen, San Jose, Calif.), cleaved PARP (BD Pharmingen), cytokeratin 19 (Abcam), cytokeratin 18 (Abeam, Cambridge, Mass.), androgen receptor (Sigma-Aldrich), prostate specific membrane antigen (Abeam), prostate specific antigen (Epitomics, Burlingame, Calif.), pan-HLA class I (Abeam), DKK1 (Orbigen, BioCarta LLC, San Diego, Calif.), activated β-Catenin (Millipore, Billerica, Mass.), β-Catenin (BD Transduction), activated Notch2 (Abeam), PTCH (Abcam), Gli1 (Santa Cruz Antibody, Santa Cruz, Calif.), Gli2 (Abeam), and β-Actin (Sigma-Aldrich) were used in immunoblot assays using standard procedures. Protein expression was quantified by comparing band expression using Quantity One software (Bio-Rad, Hercules, Calif.).
Immunofluorescence analyses were conducted on prostate cancer cell lines and formalin fixed paraffin-embedded tissue sections from human cancers and tumor xenografts. Primary antibodies included a combination of cytokeratin 19 and 18 (Abcam), pan-HLA class I (Abcam), green fluorescence protein (Abcam) and the following transcription factors: active β-Catenin (Millipore), activated Notch2 (Abcam), Gli1 (Santa Cruz), Gli2 (Abeam) and androgen receptor (DAKO, Fort Collins, Colo.). Secondary antibodies used were Alexa Fluor® 594 (Invitrogen) and Alexa Fluor® 488 (Invitrogen). Prostate cancer cells (105) were plated in 35 mm culture dishes and 24 hours later stained by standard immunofluorescence procedures. Tissue sections (5 μm) were deparaffinized and submitted to standard peroxidase based immunohistochemistry and immunofluorescence procedures. Quantification of the expression of cytokeratins, HLA class I antigen, transcription factors and androgen receptor was performed by evaluating tumoral cells. Percentage of positive and negative cells was determined in 10 high power fields.
CK19 gene promoter region was amplified from DU145 cells genomic DNA by PCR with specific primer sets (Fw 5′-AACGCATGCTTTGGGGGGATG-3′ (SEQ ID NO: 1) and Rv 5′-TCCCCCTTTACTCGGCCCCCAC-3′ (SEQ ID NO: 2)) as described previously (Tripathi et al., 2005. The PCR products were digested with Ase I and Hind III and cloned into pEGFPN1 vector (Clontech, Mountain View, Calif.) previously digested with the same enzymes. As a result, the CMV promoter was removed from the original vector and the GFP expression was under control of the CK19 promoter. The final construct was confirmed by digestion and sequencing analysis. DU145 cells were transfected with pCK19-GFP construct using Lipofectamine Plus 2000 (Invitrogen). After 24 hours, medium was replaced with fresh medium and stably expressing cells selected in the presence of G418 (Invitrogen). Positive clones were confirmed by direct microscopy and immunofluorescence and also by PCR amplification of GFP coding region using specific primers (Fw 5′-TTCCTGCGTTATCCCCTGATTC-3′ (SEQ ID NO: 3) and Rv 5′-GCTCCTCCGGCCCTTGCTCACCAT-3′ (SEQ ID NO: 4)).
Time-lapse videomicroscopy was used to assess asymmetrical cell division and Docetaxel subpopulation sensitivity of DU145 cells stably transfected with the pCK19-GFP promoter. Cells growing in 6-well plates at low confluence were placed in the stage inside an incubator chamber at 37° C., 50% humidity and in an atmosphere of 5% CO2. Unattended time-lapse movies of randomly chosen GFP+ and GFP− DU145 cells were performed with a Nikon Eclipse Ti inverted microscope. NIS Elements AR (Nikon Inc., Melville, N.Y.) software was used to collect and process data. Imaging was performed using a 10× objective and images were captured using 200-ms exposure times for GFP and 20-ms for bright field every 30 minutes.
Flow cytometry analysis of subpopulations of prostate cancer cells were carried out following standard procedures. Intracellular CK19 and CK18 expression was performed in single-cell suspensions fixed with 70% ethanol, whereas the expression of cell surface HLA class I and GFP was determined in fresh cell samples (without fixation). Primary antibodies against CK19 (Abeam), CK18 (Abeam), HLA class I (Abcam), HLA class I conjugated to phycoerythrin (Abeam) and GFP (Abcam) were used. Secondary antibodies, when used, corresponded to Alexa Fluor® 594 (Invitrogen) and Alexa Fluor® 488 (Invitrogen). Samples were acquired with a FACscan Flow Cytometer (BD Biosciences) and analyzed with a CellQuest Pro software (BD Biosciences). A minimum of 104 cells were measured per sample.
Animal use and care was in strict compliance with institutional guidelines established by the University of Columbia, Institutional Animal Care and Use Committee. Xenograft experiments were performed with 5-6 weeks old mice (NOD.CB17-Prkdcscid) obtained from Jackson Laboratories as recipients.
Formalin-fixed paraffin-embedded human primary and metastatic prostate cancer tissue samples were provided by the tumor bank of Columbia University Cancer Center. All samples were collected under informed consent and under the supervision of the Columbia University Medical Center Institutional Review Board. Tissue sections with cancer were selected by reviewing Hematoxylin & Eosin (H&E) stained slides.
Tumour Initiating Capacity of Cancer Cells from Prostate Cell Lines and Fresh Human Samples
To compare the tumor initiating capacity of Docetaxel sensitive parental cells and Docetaxel resistant cells, GFP positive and GFP negative sorted cells, or HLA-positive and HLA-negative sorted cells, various numbers of cells (e.g. 10, 102, 103, 104) were subcutaneously injected in 200 μl of medium:Matrigel (1:1) into male mice. GFP cell subpopulations of prostate cancer cells were sorted following standard procedures. For HLA class I cell isolation, single suspensions of fresh cells where blocked with PBS+FBS 5% and stained with an HLA class I antibody directly conjugated to phycoerythrin (Abcam). To assess the tumor initiating capacity of human cancer cells from fresh tumor tissue samples, portions of tumors were obtained from patients who underwent surgical procedures at Columbia University medical Center through an Institutional Review Board approved protocol. Forty-eight primary prostate cancers, 10 primary colon cancers, 10 primary lung cancers, 12 primary breast cancers and 18 primary bladder cancers were processed. Specimens were mechanically dissociated and filtered to obtain a single-cell suspension and exposed to red cell lysis buffer (Sigma-Aldrich) to remove red blood cells. Cells were stained with directly conjugated fluorescent antibodies to human CD45 (Abcam), human CD31 (eBiosciences) and human HLA-class I (Abcam). For xenograft tumors, primary fluorescent conjugated antibodies to mouse CD45 (eBiosciences), mouse CD31 (Biolegend, San Diego, Calif.) and human HLA-class I (Abcam) were used to select live human cancer cells. Cells were suspended in 10 μg/ml DAPI to label dead cells and sorted on FACSAria Cell Sorting System (BD Biosciences). Different dilutions (10, 100 and 1,000 injected cells) of human prostate cancer HLA sorted cells (HLA class I-negative and HLA class I-positive) and unsorted cells, and 100 HLA sorted cells from other human cancer samples (primary injections) and derived xenografts (secondary injections) were injected into NOD/SCID mice. Four mice for each sorted cell population and cell dilution were injected. Four injections were performed in each mouse for sorted cells, two in the upper flanks for HLA class I-negative cells and two in the lower flanks for HLA class I-positive cells. Unsorted cells from each tumour specimen were also injected in NOD/SCID mice. Secondary injections of HLA sorted cells were performed from tumours generated from HLA class I-negative sorted cells and the rarely observed tumours originated from the HLA class I-positive fraction of cells. Tumour initiation was measured by tumor incidence (number of tumors/number of injections) and latency (time from injection to first tumor palpability). Tumour formation was evaluated regularly by palpation of injection sites. In cases where a tumor became palpable at only one injection site, that tumor was surgically removed to allow continued evaluation of other injection sites. Mice were monitored for up to 36 weeks. Animals with no sign of tumor burden were also examined on necropsy to confirm that there was no tumor development. Tumors harvested were fixed in formalin, and paraffin sections were made for H&E staining and immunofluorescence studies when necessary.
The in vitro effects of Notch and Hedgehog inhibitors on HLA-negative and HLA-positive sorted cell lines were analyzed by cell cycle and colony formation assays (described above). Cells were exposed to vehicle solution (Control), dexamethasone (1 μM), Cyclopamine (1 μM), Compound E (1 μM) and a dual (e.g. dexamethasone plus Cyclopamine) or triple (dexamethasone plus Cyclopamine plus Compound E) combination of the drugs.
To analyze whether the inhibition of these developmental pathways could affect the tumor initiating capacity of the cancer stem cells in vivo, 103 HLA-negative sorted cells from cell lines and human prostate tumor xenografts were inoculated subcutaneously into NOD/SCUD mice. Mice were treated with vehicle solution (Control), dexamethasone (15 mg/kg/ip. daily), Cyclopamine (50 μg/kg/sc daily) plus dexamethasone, DBZ (10 μM/kg/ip. daily) plus dexamethasone or a combination of the 3 drugs. Dexamethasone and Cyclopamine were administered continuously; however, DBZ was administered daily (days 1 to 15 every 4 weeks) in order to avoid gut toxicity. For the in vivo cell lines studies, three independent experiments in 8 mice for treatment arm (e.g., Cyclopamine) were performed, whereas for the human prostate tumours 8 mice were included for each treatment arm. Mice were monitored every day until tumors formed. Animals were sacrificed if they showed any evidence of distress or if they lost more than 20% of their original body weight. Generated tumors were harvested and histologically confirmed.
Regarding studies on differentiation, the expression of cytokeratins (CKs) as epithelial markers was assessed, as well as prostate-related biomarkers, including AR, PSA and PSMA. CKs have been previously reported to be specific for human differentiated epithelial cells, playing a role in the maintenance of cellular integrity while also functioning in signal transduction and cellular differentiation processes. Low molecular weight CKs (e.g., CK18, and CK19) are specifically expressed in luminal normal human prostate cells and prostate cancer, whereas high molecular weight CKs (e.g., CK5 and CK10) are identified in basal normal prostate cells and rarely observed in cancer cell populations. In the model of the present invention, Docetaxel resistant cells showed a significant decrease in both gene transcription and protein expression of low molecular weight CKs. DU145-DR cells showed a 6.25 and 16.6 fold decrease in the protein expression of CKs 19 and 18 respectively. Similarly, 22RV1-DR showed a 14.3 and 6/fold decrease in such CKs when quantified and compared to the sensitive parental cells. Immunofluorescence staining of CK19 and CK18 confirmed their decreased expression in the Docetaxel resistant cells (
Regarding mechanisms of immune evasion, it was found that Docetaxel resistant cells showed deregulation of MHC class I molecules. In this context, previous work from our group already reported the identification of MHC class I antigens negative tumour cell subpopulations in human primary and metastatic carcinomas (Cordon-Cardo, et al., 1991). In the present study, it was found that MHC class I antigens, which are critical for efficient antigen presentation to cytotoxic T lymphocytes and subsequent tumour cell lysis, were down-regulated at both gene transcription and protein level (
Natural killer (NK) ligands gene expression that were deregulated in Docetaxel resistant cells (DU145-DR and 22RV1-DR) when compared to their parental sensitive cells (DU145 and 22RV1) are summarized in Table 9. Statistical analysis of the mean expression average difference of genes, which show ≧2 fold change based on a logarithmic normalization, was done using a t-test between matched sensitive and resistant cells. There is a decrease in the transcription levels of most NK ligands genes.
Regarding studies on stem cell phenotype, the identified deregulated expression of WNT/β-catenin was characterized, Notch and Hedgehog, which have been implicated in self-renewal and differentiation of progenitor cells (Katoh et al., 2007; McDonald et al., 2006; van den Brink et al., 2004; Radtke et al., 2006; Leong et al., 2008; and Grigoryan et al., 2008). In the prostate, these signaling pathways have been reported to play essential roles in developmental patterning, epithelial regeneration, and prostate cancer tumourigenesis (Wang et al., 2006; Karhadkar et al., 2004). Docetaxel resistant cells showed a significant decrease in both gene transcript and protein levels of Dickkopf-1 (DKK1), a well known inhibitor of the WNT/β-catenin signaling network. This decrease in DKK1 expression was linked to an increase in the expression of de-phosphorylated (active) β-catenin, which is the major key effector of WNT signaling. Immunofluorescence analyses demonstrated that parental Docetaxel sensitive cells displayed a membranous expression of β-catenin, associated with its function as an adhesion molecule, whereas Docetaxel resistant cells showed a pronounced nuclear localization of this protein (
Furthermore, it was observed that the reversibility of the resistant phenomenon for both DU145-DR and 22RV1-DR cells was linked to an increase in the expression of differentiation markers. Docetaxel reversed resistant cells (cultured without the drug during 12 weeks) showed higher protein expression levels of low molecular weight cytokeratins (CK19 and CK18) and HLA-class I when compared to Docetaxel acquired resistant cells, achieving levels similar to those observed in parental sensitive cells (
DU145 parental cells were stably transfected with a plasmid containing the promoter of CK19 driving the expression of the green fluorescence protein (GFP) (
In order to further confirm that HLA-class I expression can be used as a cell surface marker that identifies cells with the cancer stem cell functional property of tumour initiation, parental cell lines DU145 and 22RV1 were sorted for HLA-class I and their tumour initiating capacity tested in NOD/SCID mice (
In order to address the clonability capacity of HLA class I sorted cells from DU145 and 22RV1 parental cell lines, dilution colony formation assays were performed. HLA class I-negative cells displayed a statistically significant higher clonability than HLA class I-positive cells. Specifically, HLA class I-negative sorted cells from DU145 generated colonies in 31.6±7.5%, 17.1±3.6% and 22.0±4.9% when 10, 100 and 1000 cells were plated, respectively. In contrast, HLA class I-positive cells generated colonies in 5.0±8.3%, 8.0±3.3% and 5.5±1.5% when 10, 100 and 1000 cells were plated (
Immunohistochemical studies of metastases (n=20) and matched primary (n=6) human prostate cancer tissues revealed that all specimens contained scattered subpopulations of CK-negative (CK18 and CK19) tumour cells, accounting for 0.05% to 0.3% and 0.4% to 1.8% of all tumour cells in primary and metastatic lesions, respectively (
Experimental data is expressed as means±SD. Statistical analysis by Student's t-test was performed. Values were considered statistically significant at p≦0.05.
Based on the functional chemoresistance and phenotipical characteristics of the CSCs disclosed above, an in vitro high throughput assay was designed to screen compounds targeting this CSC population, and to determine whether CSCs are either selectively killed or differentiated. Implementation of this assay permits testing of a wide variety of drugs and facilitates the identification of compounds with a putative CSC inhibitory effect.
The in vitro high throughput assay consists of the seeding of the above mentioned CSCs into 96 well plates. Cells are then exposed to the candidate agents, e.g., drugs, of interest, and readouts are obtained at desired time points (e.g., 24 hours, 72 hours, etc). Readouts consist in comparing the percentage of cells that express the CSC phenotype (e.g., lack of HLA, CD24, etc) in treated versus untreated populations by flow cytometry analysis. In parallel, cell viability assays, which compare the percentage of viable (“surviving”) cells between treated and untreated cells, are also performed. Results are interpreted as follows: candidate agents, e.g., drugs, that target the CSC population should inhibit the growth or kill chemoresistant cells which lack CSCs. Four possible results may be obtained with this assay: outcome 1, a decrease in the percentage of CSCs with a decrease in cell viability; outcome 2, a decrease in CSC without a decrease in cell viability; outcome 3, an increase in CSC populations with a decrease in cell viability; and outcome 4, no decrease in either the percentage of CSC nor in cell viability. Outcome 1 may be interpreted that the CSC population has been targeted by the candidate agent, e.g., drug. Outcome 2 may be interpreted that the CSC population has been differentiated. Outcome 3 may be interpreted that neoplastic cells, but not the CSC population, has been targeted by the candidate agent, e.g., drug. Outcome 4 may be interpreted that no effect is produced by the candidate agent, e.g., drug, in either CSC or neoplastic differentiated cells.
Candidate agents, e.g., drugs that produce CSC depletion (outcome 1) or CSC differentiation (outcome 2) would be considered for further experimentation and characterization by means of standard in vitro and in viva assays. Briefly, these assays aimed at confirming and validating early screening results, include: a) in vitro—apoptotic methods (e.g., annexin V), colony formation, and differentiation studies based on their correponding phenotypes (e.g., flow cytometry and immunohistochemical analyses); and b) in vivo—dilutional tumor initiation assays in immunocompromised mice.
This high throughput screen is validated using the Notch and Hedgehog inhibitors disclosed above.
All documents cited in this application are hereby incorporated by reference as if recited in full herein.
Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/30103 | 3/22/2012 | WO | 00 | 1/31/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61467265 | Mar 2011 | US |
