WO2008090355A2

WO2008090355A2 - Inhibitors of stem cells markers

Info

Publication number: WO2008090355A2
Application number: PCT/GB2008/000259
Authority: WO
Inventors: Richard Birnie; Norman Maitland; Roshna Mistry
Original assignee: Procure Therapeutics Limited
Priority date: 2007-01-24
Filing date: 2008-01-24
Publication date: 2008-07-31
Also published as: GB0701321D0; WO2008090355A3

Abstract

The invention relates to agents that target cancer stem cell specific gene products and includes medicaments and methods to treat cancer, in particular prostate cancer.

Description

The invention relates to agents that target cancer stem cell specific gene products.

Prostate cancer can be relatively harmless or extremely aggressive. Some prostate tumours are slow growing and cause few clinical symptoms. Aggressive prostate tumours spread rapidly to the lymph nodes and other organs, especially bone. It is known that the growth of prostate cancer can be inhibited by blocking the supply of male hormones such as testosterone. However, prostate cancers eventually develop and become independent of male sex hormones (i.e. they become androgen-independent prostate cancer cells). These cells are linked with aggressive, malignant prostate cancer.

Metastatic prostate cancers predominantly move to bone or lymph nodes and are treated by reducing the production of androgens by blocking androgen production by the adrenal glands and testis. This treatment is only effective for a short period of time as the metastatic lesions become androgen independent and grow uncontrollably. The presence of androgen independent prostate cancer cells means that this treatment regime is no longer effective and further intervention is required to control the progress of the disease. A similar response is seen to chemotherapeutic and radiotherapy treatments. As a result, metastatic prostate cancer remains an incurable disease by current treatment strategies. There is therefore a continual need to identify new therapeutic targets to provide new treatments for prostate cancer and to provide models for testing the efficacy of agents against the progression of prostate cancer.

In our earlier application WO2005/089043 we describe a population of prostate stem cells that express CD133 antigen, a haematopoietic stem cell marker. CD133 cells are restricted to the α₂βi^hl population (the receptor for type I collagen) and are located in the basal layer, often at the base of a budding region or branching point of the prostatic ducts (Fig. 1A). α₂βi^hl /CD133⁺ cells exhibit two important attributes of epithelial stem cells: they possess a high in vitro proliferative potential (Fig. 1 B) and can reconstitute prostatic-like acini in immunocompromised male nude mice (Fig. 1C). Moreover, our co- pending application PCT application PCT/GB2006/002658 discloses gene expression microarray analysis of CD133 positive stem cells and compared expression of genes between CD133 positive stem cells and CD133 positive cancer stem cells. The array analysis identified a large number of genes that show differential expression, for example growth factors and growth factor receptors, genes that encode proteins involved in extracellular matrix formation, transcription factors and transcription factor related genes and genes involved in DNA replication and repair..

Cancer stem cells from a number of different tumour types have been identified as expressing the CD133 antigen. For example brain tumours (see Identification of a Cancer Stem Cell in Human Brain Tumors Sheila K. Singh, Ian D. Clarke, Mizuhiko Terasaki, Victoria E. Bonn, Cynthia Hawkins, Jeremy Squire and Peter B. Dirks Cancer Research 63: 5821-5828 (2003); colon tumours (see CA. O'Brien et al., "A human colon cancer cell capable of initiating tumour growth in immunodeficient mice," Nature, 445: 106-110 (2007) and L. Ricci-Vitiani et al., "Identification and expansion of human colon- cancer-initiating cells," Nature, 445: 111-115. (2007); and lung tumours (see, CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer. Hilbe W, Dirnhofer S, Oberwasserlechner F, Schmid T, Gunsilius E, Hilbe G, WoII E, Kahler CM. J Clin Pathol. 2004 Sep; 57(9):965-9).

This disclosure relates to the control of tumour initiation, progression and metastasis by the administration of agents that inhibit the activity of genes that are differentially expressed in CD133 positive cancer stem cells. The array data reveals that a large number of pro-inflammatory genes are differentially expressed in CD133 positive cancer stem cells.

Inflammation is a complex reaction of the body responding to damage of its cells and vascularised tissues. The damaged sites are susceptible to infiltration by a multitude of pathogens including viruses, bacteria, fungi, and protozoan and metazoan parasites, as well as cancerous cells and other harmful agents and so the animal defends itself by initiating an inflammatory reaction at the damaged site.

The inflammatory reaction is phylogenetically and ontogenetically the oldest defence mechanism and both the innate and adaptive immune systems in vertebrates are triggered to destroy the infectious agent(s) or cancer cell. When a tissue has been traumatised, for example, by injury or surgery, and is thus susceptible to infection, three key steps in the inflammatory response are initiated; (1) vasodilation, which enables an increased blood supply to the traumatised tissue; (2), increased capillary permeability caused by retraction of the endothelial cells allowing soluble mediators of immunity to reach the site of inflammation; and (3) migration of leukocytes (neutrophils; monocytes and lymphocytes) out of the capillaries into the surrounding tissues. The development of inflammatory reactions is controlled in part by pro-inflammatory cytokines (e.g. interleukin-1 , tumour necrosis factor alpha); by lipid mediators released from different cells (e.g. prostaglandins and leukotrienes); by cell-derived vasoactive mediators released from mast cells, basophils and platelets (e.g. arachidonic acid metabolites; platelet activating factors amines: serotonin, histamine; endothelins) and by plasma-derived vasoactive mediators (e.g. kinins and components of the complement, coagulation and fibrinolytic cascades).

As noted above the inflammatory response can play a role in tumour suppression by directing an immune response to the tumour. However, there is evidence that an immune response appears to stimulate tumour development. Indeed there is evidence that inflammatory diseases such as ulcerative colitis and Crohn's disease predispose the sufferer to cancers of the intestinal tract.

Inflammation functions at all three stages of tumour development: initiation, progression and metastasis. At initiation it contributes by inducing the release of a variety of cytokines and chemokines that promote the release of inflammatory cells and factors around the developing tumour. The role of inflammation in metastasis is less well defined. Soluble mediators produced by tumour associated leucocytes promote cell motility, angiogenesis and extravasation of cancer cells to a secondary tumour. Once established at a secondary site the inflammatory response stimulates tumour cell growth and establishment. Although there is a strong correlation between chronic inflammation and cancer the genes involved in this have not all been discovered.

In addition to pro-inflammatory genes disclosed in PCT/GB2006/002658 a number of genes unrelated to inflammation also show differential expression. These genes encode proteins with various functions, for example in signal transduction, cell motility and protein degradation. The present disclosure also relates to some of these genes.

According to an aspect of the invention there is provided an agent for use in the manufacture of a medicament that inhibits the activity of genes, either directly or indirectly, that are differentially expressed by CD133 positive cancer stem cells for the treatment of cancer.

As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.

In a preferred embodiment of the invention said cancer is prostate cancer.

In a preferred embodiment of the invention prostate cancer is primary prostate cancer.

In an alternative preferred embodiment of the invention prostate cancer is metastatic prostate cancer; in particular bone or lymph node metastatic cancer.

In a preferred embodiment of the invention said agent inhibits the activity of a polypeptide encoded by a pro-inflammatory gene.

In a further preferred embodiment of the invention said pro-inflammatory gene is interleukin 9.

In a preferred embodiment of the invention said agent is an aminosterol compound. Preferably said aminosterol is squalamine. Further examples of aminosterol compounds can be found in US08/290,826, US08/416, 883, US08/478,763, US08/483.059, US08/483,057, US08/479.457, US08/475.572, US08/476.855, US08/474, 799 and US08/487, 443 each of which is incorporated by reference in their entirety.

In a further preferred embodiment of the invention said pro-inflammatory gene is a phosphodiesterase.

In a preferred embodiment of the invention said phosphodiesterase is a PDE 4 phosphodiesterase.

In a preferred embodiment of the invention said PDE4 phosphodiesterase is selected from the group consisting of: PDE4C, PDE4A4 and PDE4B1.

In a preferred embodiment of the invention said anti-inflammatory agent is selected from the group consisting of: rolipram, cilomilast, roflumilast, BAY 19-8004, CI-1044, 7- methoxybenzofuran-4-carboxamides, C-3885, arophylline, T-2585, YM-976, phtalazine derivatives (compound 11), V-11294A, KW-4490, cipamfylline, mesopram, CC-7075, CC-7085 and piclamist.

In a preferred embodiment of the invention said agent is roflumilast comprising the structure:

In a further preferred embodiment of the invention said agent is cilomilast (ariflo) comprising the structure: Ariflo

In an alternative preferred embodiment of the invention said gene encodes a kinesin; preferably kinesin 2.

In a preferred embodiment of the invention said agent is an adociasulfate; preferably adociasulfate 2.

In a further preferred embodiment of the invention said gene encodes a Rho kinase; preferably said gene is ROCK 1 or ROCK 2.

In a preferred embodiment of the invention said agent is fasudil.

In a further preferred embodiment of the invention said agent is fasudil-OH.

In a yet further preferred embodiment said gene encodes pappalysin 1 (PAPPA).

In a preferred embodiment of the invention said agent is the polypeptide bikunin.

In an alternative preferred embodiment of the invention said agent is the polypeptide eosinophil major basic protein.

In a further preferred embodiment of the invention said gene encodes an ubiquitin specific peptidase.

In a preferred embodiment of the invention said peptidase is USP40. In a preferred embodiment of the invention said agent is selected from the group consisting of: esomerprazole, 94052 1234, ethinyestradiol/drospirenone or fuzeon.

In an alternative preferred embodiment of the invention said peptidase is USP34.

In a preferred embodiment of the invention said agent is 94052 1234.

In an alternative preferred embodiment of the invention said peptidase is USP15.

In a preferred embodiment of the invention said agent is selected from the group consisting of: iloprost, etiricoxib, lansoprazole, valganciclovir, bicalutaminde, ethinyestradiol/drospirenone.

In a further preferred embodiment of the invention said gene encodes a thymidine kinase protein; preferably TK2.

In a preferred embodiment of the invention said agent is zidovudine.

In a preferred embodiment of the invention said gene encodes presenilin.

In a preferred embodiment of the invention said agent is r-2 fluoro-alpha methyl 4 biphenyl acetic acid.

In an alternative preferred embodiment of the invention said agent is the humanized monoclonal antibody bapineuzumab.

In an alternative preferred embodiment of the invention said agent is a v secretase inhibitor, for example see Sigma product S2188 which is incorporated by reference.

In a further preferred embodiment of the invention said agent is hydroxylvaleryl monobenzocaprolactam

According to a further aspect of the invention there is provided a pharmaceutical composition comprising at least one agent that inhibits the activity of genes, either directly or indirectly, that are differentially expressed by CD133 positive cancer stem cells and at least one additional therapeutic agent effective in the treatment of cancer. - In a preferred embodiment of the invention said cancer is prostate cancer.

Preferably said therapeutic agent is a chemotherapeutic agent.

Preferably said agent is selected from the group consisting of: cisplatin; carboplatin; cyclosphosphamide; melphalan; carmusline; methotrexate; 5-fluorouracil; cytarabine; mercaptopurine; daunorubicin; doxorubicin; epirubicin; vinblastine; vincristine; dactinomycin; mitomycin C; taxol; L-asparaginase; G-CSF; etoposide; colchicine; derferoxamine mesylate; and camptothecin.

In a further preferred embodiment of the invention said agent is selected from the group consisting of: docetaxel, mitoxantrone, epirubicin and paclitaxel.

When administered, the compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives and compatible carriers.

The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.

The compositions of the invention are administered in effective amounts. An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of treating a particular disease, such as cancer, the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods.

Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of agent for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by measuring the physiological effects of the composition, such as regression of a tumour, decrease of disease symptoms, modulation of apoptosis, etc.

The doses of agent administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

Other protocols for the administration of agents will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration (e.g., intra-tumoural) and the like vary from the foregoing. Administration of compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

Compositions may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of agent, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

According to a further aspect of the invention there is provided a method to treat cancer comprising administering an agent that inhibits the activity of genes, either directly or indirectly, that are differentially expressed by CD133 positive cancer stem cells.

In a preferred method of the invention said cancer is prostate cancer.

In a preferred method of the invention prostate cancer is primary prostate cancer.

In an alternative preferred method of the invention prostate cancer is metastatic prostate cancer; in particular bone or lymph node metastatic cancer.

In a preferred method of the invention said agent inhibits the activity of a polypeptide encoded by a pro-inflammatory gene.

In a further preferred method of the invention said pro-inflammatory gene is interleukin 9.

In a preferred method of the invention said agent is an aminosterol compound. Preferably said aminosterol is squalamine.

In a further preferred method of the invention said pro-inflammatory gene is a phosphodiesterase. In a preferred method of the invention said phosphodiesterase is a PDE 4 phosphodiesterase.

In a preferred method of the invention said PDE4 phosphodiesterase is selected from the group consisting of: PDE4C, PDE4A4 and PDE4B1.

In a preferred method of the invention said anti-inflammatory agent is selected from the group consisting of: rolipram, cilomilast, roflumilast, BAY 19-8004, CI-1044, 7- methoxybenzofuran-4-carboxamides, C-3885, arophylline, T-2585, YM-976, phtalazine derivatives (compound 11), V-11294A, KW-4490, cipamfylline, mesopram, CC-7075, CC-7085 and piclamist.

In a preferred method of the invention said agent is roflumilast comprising the structure:

In a further preferred method of the invention said agent is cilomilast (ariflo) comprising the structure:

Ariflo

In an alternative preferred method of the invention said gene encodes a kinesin; preferably kinesin 2.

In a preferred method of the invention said agent is a adociasulfate; preferably adociasulfate 2.

In a further preferred method of the invention said gene encodes a Rho kinase; preferably said gene is ROCK 1 or ROCK 2.

In a preferred method of the invention said agent is fasudil.

In a further preferred method of the invention said agent is fasudil-OH.

In a yet further preferred method said gene encodes pappalysin 1.

In a preferred method of the invention said agent is the polypeptide bikunin.

In a further preferred method of the invention said gene encodes an ubiquitin specific peptidase.

In a preferred method of the invention said peptidase is USP40.

In a preferred method of the invention said agent is selected from the group consisting of: esomerprazole, 94052 1234, ethinyestradiol/drospirenone or fuzeon. In an alternative preferred method of the invention said peptidase is USP34.

In a preferred method of the invention said agent is 94052 1234.

In an alternative preferred method of the invention said peptidase is USP15.

In a preferred method of the invention said agent is selected from the group consisting of: iloprost, etiricoxib, lansoprazole, valganciclovir, bicalutaminde, ethinyestradiol/drospirenone.

In a further preferred method of the invention said gene encodes a thymidine kinase protein; preferably TK2.

In a preferred method of the invention said agent is zidovudine.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 : Verification of CD133 as a stem cell marker of prostatic epithelia: 1A: A paraffin section of prostatic acini labelled with the nuclear stain DAPI (Blue) and anti- CD133 directly conjugated to PE (Red). 1B: Basal cells with the phenotype α₂βi^hi /CD133⁺ have a higher colony forming efficiency (CFE) than α₂β ^|OW/CD133^". (CFE) was calculated as the number of colonies formed per number of selected cells x100%. CFEs are expressed as the ratio of the control CFE. Results show means D s.e.m of four experiments. 1C. Xenografts of prostate acini formed by transplantation of α₂βi^hl /CD133⁺ basal cells stained with (A) Haematoxylin and Eosin, (B) 34βE12, (C) anti-K18, (D) anti-PAP (E) Anti-androgen receptor. Bar 40 Dm;

Figure 2: Characterisation of tumour 'stem' cells from a lymph node metastasis of the prostate (LNMP). 2A. Tumour cells selected on the basis of α₂β-ι/CD133 differentiate in culture. 2B. Invasion assay activity Prostate CSCs (First 4 lanes) are 2-3 fold more invasive in comparison to PC3M Normal SC (PE583) BPH cultures (n=3) and an immortalised prostate epithelial cell line, PNTIa (Collins et al (2005) Cancer Res. 65, 10946-51;

Figure 3: Primary (dark boxes) and secondary (unfilled boxes) colony forming efficiency of prostate cancer stem, amplifying and luminal cell populations; and

Materials and Methods

Genotype of isolated tumour stem cells

Using a combination of microsatellite markers associated with sporadic prostate cancer (8p 10q 16p, Fig 3) we can determine whether the isolated HEA⁺/CD44⁺/α₂β₁ ^hi /CD133⁺ cells display loss of heterozygosity patterns characteristic of prostate tumours in comparison to blood lymphocyte DNA from the same patient. The analysis is carried out on a microsampling of cultures with 3MM paper and fluorescently labelled PCR primers

(Macintosh et al., 1998). This will enable us to discriminate between normal and cancer cells and determine whether stem cells are indeed targets for transforming events.

Proliferative, differentiative and malignant potential of putative cancer stem cells

Distinct populations of tumour cells are isolated and their proliferative, differentiative and malignant potential determined in vitro and in vivo. The following populations (HEA⁺/CD44^" (luminal cells), HEA⁺/CD44⁺ (basal cells), HEA⁺/CD44⁺/ C^₁'⁰" /CD133^" (transit cells), HEA⁺/CD44⁺/ α₂βi^hl /CD133⁺ (stem cells) are isolated and compared with the unsorted tumour population. Assays for testing agent activity

A number of assays are available to test the efficacy of agents disclosed in the present application.

Colony forming efficiency (CFE): anchorage independent and anchorage- dependent growth

The transforming potential of distinct populations (as above) of cancer cells (anchorage independence) is measured by their ability to form colonies in soft agar. Individual colonies are counted after 21 days using an inverted microscope. Comparisons are made of CFE and colony size.

Morphogenesis in gels of reconstituted basement membrane matrix

We have determined the potential of tumour stem cells and their progenitors to undergo glandular morphogenesis in reconstituted basement membrane (e.g. Matrigel). We have demonstrated that normal basal cells can undergo glandular morphogenesis when grown in a collagen based matrix, (e.g. Matrigel) with stroma, in the presence of androgens. Spheroids are generated which are architecturally and phenotypically similar to in vivo acini and are often branched alveolar-and duct like (Lang ef a/., 2001) In contrast, cancer cells often form large aggregates of spindle-shaped cells with no obvious organisation. Nonetheless, the structures will often contain cells that show some degree of differentiation and can be compared to the original tumour.

Invasion assays

The ability of these stem cells to migrate across Matrigel is determined by the modified Boyden-chamber method (Albini ef a/., 1987). Migration rates will be evaluated using time-lapse near-confocal microscopy, using cells labelled with EGFP or its red equivalents. We have generated prostate epithelium expressing low levels of EGFP. Recombinant Antiviruses have the capacity to infect and fluorescently label prostate CSCs successfully infected cells are selected on the basis of resistance to blasticidin and used in motility/invasion assays. The low levels of GFP expression will be used to track invasion and motility in real time. In vivo tumourigenesis

Tumour stem cells must possess key criteria that define normal stem cells: after transplantation they must proliferate, differentiate and self-renew. To determine the ability of distinct tumour phenotypes, to colonise in vivo, grafts of stem cells, transit cells, basal cells, luminal cells and unsorted cells are introduced into the prostates of 6 to 8 week old male, immune compromised mice. The mice are treated hormonally at the time of grafting by subcutaneous implantation of sustained release testosterone pellets. The number of cells from each population that successfully engraft and initiate tumour proliferation is determined by varying the number of cells implanted. The self-renewal capacity of the distinct populations is determined by transplanting serially into secondary recipients.

Colony Forming assays in vitro to assess stem cell specificity

While conventional drug sensitivity assays measure inhibition of cell proliferation, such assays are unlikely to be successful with relatively quiescent cancer stem cells. One property of CSCs is their inherent ability to generate clonal cell growth in vitro. Amplifying and luminal cells have only moderate and low primary colony forming activity respectively, and little or no secondary colony forming ability. Therefore to assay for CSC-specific toxicity the secondary colony forming efficiency of the cell population, grown from an initial stem cell culture should be measured after treatment with potential CSC cytotoxic compounds. If secondary colony forming is inhibited, then CSCs have been removed. Secondary toxic effects on amplifying and luminal cells particularly from cancer-derived cultures are inconsequential.

Claims

1. The use of an agent in the manufacture of a medicament that inhibits the activity of genes, either directly or indirectly, that are differentially expressed by CD133 positive cancer stem cells for the treatment of cancer.

2. Use according to claim 1 wherein said cancer is prostate cancer.

3. Use according to claim 2 wherein prostate cancer is primary prostate cancer.

4. Use according to claim 2 wherein prostate cancer is metastatic prostate cancer.

5. Use according to claim 4 wherein metastatic cancer is bone metastatic cancer.

6. Use according to any of claims 1-5 wherein said gene encodes pappalysin 1.

7. Use according to claim 6 wherein said agent is the polypeptide bikunin.

8. Use according to claim 6 wherein said agent is the polypeptide eosinophil major basic protein.

9. Use according to any of claims 1-5 wherein said agent inhibits the activity of a polypeptide encoded by a pro-inflammatory gene.

10. Use according to claim 9 wherein said pro-inflammatory gene is interleukin 9.

11. Use according to claim 10 wherein said agent is an aminosterol compound.

12. Use according to claim 11 wherein said aminosterol is squalamine.

13. Use according to claim 9 wherein said pro-inflammatory gene is a phosphodiesterase.

14. Use according to claim 13 wherein said phosphodiesterase is a PDE 4 phosphodiesterase.

15. Use according to claim 14 wherein said PDE4 phosphodiesterase is selected from the group consisting of: PDE4C, PDE4A4 and PDE4B1.

16. Use according to any of claims 13-15 wherein said anti-inflammatory agent is selected from the group consisting of: rolipram, cilomilast, roflumilast, BAY 19-8004, Cl-

1044, 7-methoxybenzofuran-4-carboxamides, C-3885, arophylline, T-2585, YM-976, phtalazine derivatives (compound 11), V-11294A, KW-4490, cipamfylline, mesopram, CC-7075, CC-7085 and piclamist.

17. Use according to claim 16 wherein said agent is roflumilast comprising the structure:

18. Use according to claim 16 wherein said agent is cilomilast (ariflo) comprising the structure:

Ariflo

19. Use according to any of claims 1-5 wherein said gene encodes a kinesin;

20. Use according to claim 19 wherein said kinesin is kinesin 2.

21. Use according to claim 19 or 20 wherein said agent is a adociasulfate.

22. Use according to claim 21 wherein said agent is adociasulfate 2.

23. Use according to any of claims 1-5 wherein said gene encodes a Rho kinase.

24. Use according to claim 23 wherein said Rho kinase is ROCK 1 or ROCK 2.

25. Use according to claim 23 or 24 wherein said agent is fasudil.

26. Use according to claim 23 or 24 wherein said agent is fasudil-OH.

27. Use according to any of claims 1-5 wherein said gene encodes an ubiquitin specific peptidase.

28. Use according to claim 27 wherein said peptidase is USP40.

29. Use according to claim 28 wherein said agent is selected from the group consisting of: esomerprazole, 94052 1234, ethinyestradiol/drospirenone or fuzeon.

30. Use according to claim 29 wherein said peptidase is USP34.

31. Use according to claim 30 wherein said agent is 94052 1234.

32. Use according to claim 27 wherein said peptidase is USP15.

33. Use according to claim 32 wherein said agent is selected from the group consisting of: iloprost, etiricoxib, lansoprazole, valganciclovir, bicalutaminde, ethinyestradiol/drospirenone.

34. Use according to any of claims 1-5 wherein said gene encodes a thymidine kinase protein; preferably TK2.

35. Use according to claim 34 wherein said agent is zidovudine.

36. Use according to any of claims 1-5 wherein said gene encodes presenilin.

37. Use according to claim 36 wherein said agent is r-2 fluoro-alpha methyl 4 biphenyl acetic acid.

38. Use according to claim 36 wherein said agent is the humanized monoclonal antibody bapineuzumab.

39. Use according to claim 36 wherein said agent is hydroxylvaleryl monobenzocaprolactam.

40. A pharmaceutical composition comprising at least one agent that inhibits the activity of genes, either directly or indirectly, that are differentially expressed by CD133 positive cancer stem cells and at least one additional therapeutic agent effective in the treatment of cancer.

41. A composition according to claim 40 wherein said cancer is prostate cancer.

42. A composition according to claim 40 or 41 wherein said therapeutic agent is a chemotherapeutic agent.

43. A composition according to any of claims 40-42 wherein said agent is selected from the group consisting of: cisplatin; carboplatin; cyclosphosphamide; melphalan; carmusline; methotrexate; 5-fluorouracil; cytarabine; mercaptopurine; daunorubicin; doxorubicin; epirubicin; vinblastine; vincristine; dactinomycin; mitomycin C; taxol; L- asparaginase; G-CSF; etoposide; colchicine; derferoxamine mesylate; and camptothecin.

44. A composition according to any of claims 40-42 wherein said agent is selected from the group consisting of: docetaxel, mitoxantrone, epirubicin and paclitaxel.