WO2012046064A2

WO2012046064A2 - Cell differentiation

Info

Publication number: WO2012046064A2
Application number: PCT/GB2011/051913
Authority: WO
Inventors: Jayant Rane; Norman Maitland; Alastair Droop
Original assignee: The University Of York
Priority date: 2010-10-07
Filing date: 2011-10-06
Publication date: 2012-04-12
Also published as: GB201016852D0; WO2012046064A3

Abstract

The disclosure relates to the expression of a combination of three or four genes the expression of which modulates the differentiation state of a stem cell, in particular a cancer stem cell.

Description

Cell Differentiation

The disclosure relates to the expression of a combination of two, three or four genes the expression of which modulates the differentiation state of a stem cell, in particular a cancer stem cell.

The term "stem cell" represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues. Stem cells can be pluripotent or multipotent. A pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism. A multipotent cell has a restricted ability to form differentiated cells and tissues. Typically adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought. A totipotent cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta). Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism and are not as such considered stem cells. Embryonic stem cells (i.e. those having the characteristic of pluripotentiality) may be principally derived from two embryonic sources. Cells isolated from the inner cell mass are termed embryonic stem (ES) cells. In the laboratory mouse, similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These would ultimately differentiate into germ cells and are referred to as embryonic germ cells (EG cells). Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types but possible differences in behaviour (e.g. with respect to imprinting) have led these cells to be distinguished from one another.

Evidence suggests that tumours are clonal and are therefore derived from a single cell. However, there are few studies that identify and characterize those cells types that are responsible for maintaining tumour cell growth. Some have searched for these so called "cancer stem cells". The concept of a cancer stem cell within a more differentiated tumour mass, as an aberrant form of normal differentiation, is now gaining acceptance over the current model of oncogenesis in which all tumour cells are equivalent both in growth and tumour-initiating capacity [Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 1977, 197: 461463; Pardal R, Clarke MF, Morrison SJ: Applying the principles of stem cell biology to cancer. Nat. Rev. Cancer 2003, 3: 895902.] For example, in leukaemia, the ability to initiate new tumour growth resides in a rare phenotypically distinct subset of tumour cells [Bonnet D, Dick J.E. Human acute myeloid leukaemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nat. Med. 1997, 3: 730737] which are defined by the expression of CD34 and CD38 surface antigens and have been termed leukaemia stem cells. Similar tumour-initiating cells have also been found in 'solid' cancers such as prostate [Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ: Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res. 2005, 65: 1094610951 ], breast [Al Hajj M, Wicha MS, BenitoHernandez A, Morrison SJ, Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100: 39833988], brain [Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB: Identification of human brain tumour initiating cells. Nature 2004, 432: 396401 ], lung [Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I., Vogel S, Crowley D, Bronson RT, Jacks T: Identification of bronchioalveolar stem cells in normal lung and lung cancer. Ce// 2005, 121 : 823-835] colon [O'Brien CA, Pollett A, Gallinger S, Dick JE: A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007, 445: 1061 10; RicciVitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R: Identification and expansion of human colon cancer initiating cells. Nature 2007, 445: 1 1 1 1 15]; and gastric cancers [Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR, Wang TC: Gastric cancer originating from bone marrow derived cells. Science 2004, 306: 15681571 ].

This disclosure relates to the identification of genes that regulate stem cell differentiation. The evidence suggests that expression of these genes in a cancer stem cell induces differentiation to a differentiated cancer cell. The disclosure relates to the modulation of expression of these genes on the phenotype of stem/differentiated cells and also as a diagnostic test for detection of early events in cancer stem cell differentiation. The genes are lipocalin 2 [LCN2], Carcinoembryonic Antigen-Related Cell Adhesion Molecule [CEACAM6], S100 Calcium-Binding Protein P [S100P] and small proline rich protein 3 LCN2 is a secreted iron delivery molecule and is expressed in epithelia including prostate epithelium. LCN2 has been implicated in differentiation of hematopoietic and epidermal system. LCN2 is up-regulated in a variety of precancerous conditions such as oesophageal dysplasia and multiple cancers such as thyroid cancer and AML. It has been suggested that LCN2 promotes invasiveness and motility in breast cancer, colon cancer and cholangiocarcinoma cell lines. LCN2 has also been suggested as a prognostic marker for colorectal cancer and ovarian cancer. It has also been widely investigated as a marker for acute and chronic renal injury. LCN2 has been proposed to have bacteriostatic action in iron-limiting conditions. It has been also implicated in epithelial to mesenchymal transition.

CEACAM6 (NCA) was sequenced by Barnett et al. in 1988 and was shown to be structurally and probably functionally distinct from its well known family member carcinoembryonic antigen [CEA]. CEACAM6 is thought to regulate haematopoietic and colonocyte differentiation. It has been suggested to play positive role in cancer progression and could be a prognostic marker for AML, colorectal cancer, breast cancer, pancreatic adenocarcinoma and ovarian cancer.

S100p regulates cytoplasmic Ca⁺⁺ levels. S100p supposedly regulates cell survival, proliferation and migration. Therefore it is proposed to be positively involved in the formation of lung cancer, intraductal papillary breast cancer, hepatocellular cancer, ovarian and pancreatic cancers. It has been reported that S100p is hypermethylated in prostate cancer contributing to prostate carcinogenesis. S100p is also been related to pancreatic differentiation.

Small Proline Rich Protein 3 [SPRR3] is part of a epidermal differentiation cluster (EDC) on chromosome 1 q. SPRR3 is associated with psoriasis and is implicated in epidermal and oesophageal epithelial differentiation.

Although the prior art teaches the association of these individual genes in various aspects of cancer initiation and metastasis there is no disclosure of the combination of expression these genes in cancer cells and therefore the combination of genes is new. According to an aspect of the invention there is provided a diagnostic method for the detection of cancer cells isolated from a subject comprising determining the expression of one or more genes selected from the group consisting of: lipocalin 2, carcinoembryonic antigen-related cell adhesion molecule, S100 calcium-binding protein P and optionally small proline rich protein 3 wherein over-expression of said gene is indicative of cancer or a predisposition to cancer in said subject.

In a preferred method of the invention said method detects expression of two or more genes; or detection of three or more genes.

In a preferred method of the invention said method detects over expression of lipocalin 2, carcinoembryonic antigen-related cell adhesion molecule and S100 calcium-binding protein P.

In a preferred method of the invention said method comprises:

i) providing an isolated biological sample to be tested;

ii) forming a preparation comprising said sample and an oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 7a, 8a, 9a or Figure 10a; a thermostable DNA polymerase, deoxynucleotide triphosphates and co- factors;

iii) providing polymerase chain reaction conditions sufficient to amplify said nucleic acid molecule;

iv) analysing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid molecule comprising a nucleotide sequence derived from Figure 7a, 8a, 9a or Figure 10a; and optionally v) comparing the amplified product with a normal matched control.

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 tuours, 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 method of the invention said cancer is a carcinoma.

In an alternative preferred method of the invention said method comprises:

i) providing an isolated biological sample to be tested; ii) forming a preparation comprising said sample and an antibody or antibodies that specifically binds one or more polypeptide^] in said sample as represented by the amino acid sequences presented in Figures 7b, 8b, 9b or 10b to form an antibody/polypeptide complex;

iii) detecting the complex or complexes so formed; and iv) comparing the expression of said polypeptide^] with a normal matched control. According to a further aspect of the invention there is provided a composition comprising one or more antisense RNA molecules wherein said antisense RNA molecule comprise a nucleotide sequence adapted to anneal to a sense nucleotide sequence derived from at least one gene represented by the sense sequence presented in Figure 7a, 8a, 9a, or 10a.

In a preferred embodiment of the invention said composition is a pharmaceutical composition.

In a preferred embodiment of the invention said composition consists essentially of one or more antisense RNA molecules and physiologically compatible excipients and/or adjuvants. In a preferred embodiment of the invention said antisense RNA molecule is derived from two of the three nucleotide sequences presented in Figures 7a, 8a, 9a or 10a. Preferably said antisense RNA molecules are derived from each of the nucleotide sequences presented in Figures 7a, 8a or 9a.

In a preferred embodiment of the invention said antisense RNA molecule is part of a siRNA or shRNA molecule.

A technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as small inhibitory or interfering RNA (siRNA), into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA molecule. The siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The siRNA molecule is typically derived from exons of the gene which is to be ablated. The mechanism of RNA interference is being elucidated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21 -29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.

In a preferred embodiment of the invention said antisense RNA molecule is between 19 nucleotides [nt] and 29nt in length. More preferably still said antisense RNA molecule is between 21 nt and 27nt in length. Preferably said antisense RNA molecule is about 21 nt in length.

In a preferred embodiment of the invention said antisense RNA consists of 21 nt.

In a preferred embodiment of the invention said antisense, siRNA or shRNA includes modified nucleotides.

The term "modified" as used herein describes a nucleic acid molecule in which; i) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide). Alternatively or preferably said linkage may be the 5' end of one nucleotide linked to the 5' end of another nucleotide or the 3' end of one nucleotide with the 3' end of another nucleotide; and/or ii) a chemical group, such as cholesterol, not normally associated with nucleic acids has been covalently attached to the double stranded nucleic acid. iii) Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.

The term "modified" also encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified nucleotides may also include 2' substituted sugars such as 2'-0-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose. Modified nucleotides are known in the art and include, by example and not by way of limitation, alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil;5- carboxymethylaminomethyl-2-thiouracil; 5 carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; l-methyladenine; 1 -methylpseudouracil; 1 - methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3- methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5- methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; β-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2 methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2- thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5— oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1 -methylguanine; 1 -methylcytosine. Modified double stranded nucleic acids also can include base analogs such as C-5 propyne modified bases (see Wagner et al., Nature Biotechnology 14:840-844, 1996).

In an alternative preferred embodiment of the invention said pharmaceutical composition includes a carrier adapted to deliver said antisense RNA to a cell or tissue.

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, compatible carriers and supplementary anti-cancer agents.

The compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time. Treatment may be topical or systemic. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal, transepithelial or intra bone marrow administration.

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. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of an agent according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient. The doses of the antisense RNA according to the invention 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.

In general, doses of antisense RNA e.g., siRNA of between 1 nM - 1 μΜ generally will be formulated and administered according to standard procedures. Preferably doses can range from 1 nM-500nM, 5nM-200nM, and 10nM-100nM. Other protocols for the administration of compositions 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 and the like vary from the foregoing. The 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' (e.g. anti-inflammatory agents such as steroids, non-steroidal anti-inflammatory 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" in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application, (e.g. liposome or immuno-liposome). 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 or as a gel. Compositions may be administered as aerosols and inhaled. 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, for example, as a solution in 1 , 3-butane diol. Among the acceptable 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. In an alternative preferred embodiment of the invention said shRNA is part of an expression vector adapted for eukaryotic expression; preferably said shRNA is operably linked to at least one promoter sequence.

In a preferred embodiment of the invention said vector is adapted by inclusion of a transcription cassette comprising a nucleic acid molecule wherein said cassette comprises the nucleotide acid sequence which is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette wherein said sense and antisense nucleic acid molecules are adapted to anneal over at least part of their length to form an shRNA.

In a preferred embodiment of the invention said cassette is provided with at least two promoters adapted to transcribe both sense and antisense strands of said nucleic acid molecule. In a further preferred embodiment of the invention said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts thereby forming an shRNA. According to a further aspect of the invention there is provided a composition comprising one or more expression vectors wherein said vector[s] are adapted for expression of at least two nucleic acid molecules comprising nucleotide sequences selected from the group consisting of:

i) nucleotide sequences represented by the sequences in Figures 7a, 8a,

9a, or 10a;

ii) nucleic acid molecules comprising a nucleotide sequence the complementary sequence of which hybridizes under stringent hybridization conditions to the sequences represented in Figures 7a, 8a, 9a or 10a wherein said adaptation is the over-expression of said nucleic acid molecules when said vectors are transfected into a cell.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize)

Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows seguences that share at least 80% identity to hybridize)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows seguences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

In a preferred embodiment of the invention said adaptation is the provision of a regulatable promoter, for example inducible, developmental or cell/tissue specific. Preferably said promoter is cancer specific.

"Promoter" is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only. Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of physiological/environmental cues.

Promoter elements also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

Adaptations also include the provision of selectable markers and autonomous replication sequences which facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors.

Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. Expression control sequences also include so-called Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number-dependent expression to linked genes when assayed as transgenic constructs. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al., Cell (1987), 51 : 975-985.

There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

The use of viruses or "viral vectors" as therapeutic agents is well known in the art. Additionally, a number of viruses are commonly used as vectors for the delivery of exogenous genes. Commonly employed vectors include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from retroviridae baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxviridae, adenoviridiae, or picornnaviridiae. Chimeric vectors may also be employed which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al. (1997) Nature Biotechnology 15:866-870). Such viral vectors may be wild-type or may be modified by recombinant DNA techniques to be replication deficient, conditionally replicating or replication competent.

Preferred vectors are derived from retroviral genomes [e.g. lentivirus].

Alternatively, said vectors are derived from baculovirus genomes.

Viral vectors may be conditionally replicating or replication competent. Conditionally replicating viral vectors are used to achieve selective expression in particular cell types while avoiding untoward broad spectrum infection. Examples of conditionally replicating vectors are described in Pennisi, E. (1996) Science 274:342-343; Russell, J. (1994) Eur. J. of Cancer 30A(8):1 165-1 171 . Additional examples of selectively replicating vectors include those vectors wherein a gene essential for replication of the virus is under control of a promoter which is active only in a particular cell type or cell state such that in the absence of expression of such gene, the virus will not replicate. Examples of such vectors are described in Henderson, et al., United States Patent No. 5,698,443 issued December 16, 1997 and Henderson, et al.; United States Patent No. 5,871 ,726 issued February 16, 1999 the entire teachings of which are herein incorporated by reference.

Additionally, the viral genome may be modified to include inducible promoters which achieve replication or expression only under certain conditions. Examples of inducible promoters are known in the scientific literature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; lida, et al. (1996) J. Virol. 70(9):6054- 6059; Hwang, et al. (1997) J. Virol 71 (9):7128-7131 ; Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al. (1997) J. Biol. Chem 272(46); 29364-29371 . According to a further aspect of the invention there is provided an isolated mammalian cell modified by transfection with a vector composition according to the invention wherein said cell expresses at least two nucleic acid molecules selected from the group consisting of:

i) nucleotide sequences represented by the sequences in Figures 7a, 8a,

9a, or 10a;

In a preferred embodiment of the invention said cell over-expresses a nucleic acid comprising a nucleotide sequence as represented in Figure 7a, 8a and 9a.

In a preferred embodiment of the invention said cell over-expresses a nucleic acid comprising a nucleotide sequence as represented in Figure 7a, 8a, 9a and 10a.

According to an alternative preferred aspect of the invention there is provided an isolated mammalian cell modified by transfection with an antisense composition according to the invention wherein the expression of at least two genes represented by the nucleotide sequences in Figures 7a, 8a, 9a or 10a are down-regulated or ablated.

In a preferred embodiment of the invention said cell is transfected with an antisense composition comprising antisense molecules designed with reference to nucleotide sequences represented in Figure 7a, 8a and 9a.

In a preferred embodiment of the invention said mammalian is a human cell. According to a further aspect of the invention there is provided an antisense composition according to the invention for use in the treatment of cancer.

According to a further aspect of the invention there is provided a vector composition according to the invention for use in the treatment of cancer. In a preferred embodiment of the invention said cancer is a carcinoma; preferably a prostate carcinoma.

According to an aspect of the invention there is provided a diagnostic method for the detection of cancer cells isolated from a subject comprising determining the expression of one or more genes selected from the group represented in table 1 wherein over- expression of said gene is indicative of cancer or a predisposition to cancer in said subject.

According to a further aspect of the invention there is provided a composition comprising one or more antisense RNA molecules wherein said antisense RNA molecule comprise a nucleotide sequence adapted to anneal to a sense nucleotide sequence derived from at least one gene represented in table 1 . According to a further aspect of the invention there is provided a composition comprising one or more expression vectors wherein said vector[s] are adapted for expression of at least two nucleic acid molecules comprising nucleotide sequences selected from nucleotide sequences represented by the genes presented in table 1 . According to a further aspect of the invention there is provided an isolated mammalian cell modified by transfection with a vector composition according to the invention wherein said cell expresses at least two nucleic acid molecules selected from the nucleotide sequences represented by the genes presented in table 1 . According to an alternative preferred aspect of the invention there is provided an isolated mammalian cell modified by transfection with an antisense composition according to the invention wherein the expression of at least two genes represented by the nucleotide sequences represented by the genes in table 1 are down-regulated or ablated. 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 illustrates differential expression of candidate gene in microarray data. The microarray data had 6 chips of gene expression in Benign stem (BS-red) cells, 6 of benign committed basal (BC-blue) cells, 12 of malignant stem (MS-green) cells and 12 of malignant committed basal (MC-violet) cells. Each dot indicates expression on an individual chip and horizontal line indicates mean. All the candidate genes are up- regulated in committed cells as compared to stem cells. CEACAM6 and SPRR3 have two probes on each chip; whereas, LCN2 and S1 OOp have one;

Figure 2 illustrates candidate gene expression in primary prostate epithelium. Candidate genes are up-regulated in committed basal (CB) cells as compared to Stem cells (SC) and transit amplifying (TA) cells;

Figure 3 illustrates candidate gene expression in prostate cell lines. Candidate genes except SPRR3 are up-regulated in prostate cell line with committed basal cell phenotype (green) than cell lines with luminal cell phenotype (blue). SPRR3 is undetectable in all the cell lines except PNT2c2;

Figure 4 illustrates a correlation of candidate gene over a large dataset. Genes correlating with S100p expression were identified in a microarray dataset with 800 published experiments containing nearly 25,000 microarray chips. In this dataset, it has been found that CEACAM6 and LCN2 correlate with S100p expression more than that of any other gene; Figure 5 illustrates promoter analysis for candidate genes. Bioinformatic analysis of promoters of candidate genes revealed that there are 40 transcription factors (TFs) that can bind to at least 3 of the genes (A & B). There are 8 common TF binding sites to all 4 of the genes (C). This list includes some strong pro-differentiation TFs such as PAX3, PAX6, PEA3, SOX9 and ARE;

Figure 6 illustrates 5-Azacytidine (Azt) and Trichostatin-A (TSA) treatment for preliminary epigenetic analysis. (A): Azt treatment of prostate cell lines showed marked increase in S100p expression after Azt treatment as opposed to control DMSO treatment implying the role of DNA methylation in the regulation of S100p expression. (B): Histone acetylation could regulate CEACAM6 expression as CEACAM6 expression is upregulated after TSA treatment significantly;

Figure 7a is the nucleotide sequence that encodes human lipocalin 2 [LCN2], Figure 7b is the amino acid sequence of LCN2;

Figure 8a is the nucleotide sequence of human Carcinoembryonic Antigen-Related Cell Adhesion Molecule [CEACAM6]; Figure 8b is the amino acid sequence of CEACAM6;

Figure 9a is the nucleotide sequence of human S100 Calcium-Binding Protein P [S100P]; Figure 9b is the amino acid sequence of S100P; and

Figure 10a is the nucleotide sequence of human small proline rich protein 2 [SPRP2] isoform 1 and isoform 2; Figure 10b is the amino acid sequence of SPRP2 isoform 1 and 2.

Materials and Methods Isolation of prostate epithelial sub-population:

Please refer to Collins, A. T., Berry, P. A., Hyde, C, Stower, M. J., and Maitland, N. J. (2005). Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65, 10946-10951 . qRT-PCR conditions:

50°C for 15 min

95 °C for 2 min

Followed by 40 cycles of (95 °C for 15s and 60 °C for 30s)

Comparison of gene expression co-relation over large microarray dataset:

A quality controlled dataset of 800 published human microarray experiments was generated in the lab.

Promoter analysis:

Genomatix Matlnspector software was used to identify transcription binding sites on candidate gene promoters.

Methyl ati on analysis with 5-Azacytidine:

1 μΜ 5-Azacytidine treatment for 96 hrs, cell media changed daily with fresh drug. Control: DMSO

Histone acetylation analysis with Trichostatin A:

0.6 μΜ Trichostatin-A treatment for 48 hrs, Control: DMSO.

Example 1

Prostate cancer (PCa) is one of the most common male cancers. Management of frequent relapses after androgen ablation is the challenge for PCa treatment. It has been suggested that conventional therapies kill differentiated cells, but undifferentiated quiescent cancer stem cells (CSCs) persist and are responsible for relapse. Identifying key genetic and epigenetic regulators of prostate stem cell (SC) differentiation to basal cells (CB) through transit amplifying (TA) cells could lead to therapeutically exploitable targets that will induce differentiation in CSCs, making them susceptible to conventional therapies.

In this study, we fractionated benign and malignant human primary prostate epithelial cultures (PPECs) (by MACS sorting) using differential a231 integrin and CD133 expression to isolate pure sub-populations. Based on several criteria:

• At least 8-fold up-regulation in CB vs. SC

• Minimal expression in SCs

· Role in stem cell differentiation, carcinogenesis

• Possible role in prostate cancer and differentiation

• Possibility of epigenetic regulation

LCN2, CEACAM6, S100p, and SPRR3 were identified as key differentially expressed genes in SC (CD133+/a231 integrinhi) vs. CB (CD133-/a231 integrinlow) by reanalysing published microarray data from our lab (Birnie et al., 2008, ArrayExpress accession ID: E-MEXP-993) with the aim of investigating epithelial differentiation in detail (Fig.1 ).

Example 2

Results obtained from candidate gene expression qRT-PCR analysis in PPECs confirmed that candidate genes are up-regulated in CB cells as compared to SC confirming microarray results. Candidate gene expression was found to be minimal in TA cells (Fig. 2). Expression analysis in cell lines revealed that candidate gene expression is down-regulated in cell lines with luminal cell (LC) phenotype that is further down in the prostate hierarchy as opposed to cell lines with basal cell phenotype (Fig. 3). This expression profile suggests that candidate genes are expressed lowly in SC and TA then high in CB cells and then again low in LC. Therefore, it seems that these genes are important for inducing and maintaining basal cell phenotype.

Example 3

Analysis of a large database of published human microarrays (-25,000 chips) showed that S100p expression correlated with LCN2 and CEACAM6 more than with any other gene (Fig. 4). This strong correlation suggests that these genes have identical function and may be regulated by a similar or common mechanism. Example 4

Promoter analysis revealed binding sites for pro-differentiation transcription factors such as AR, BNC1 , and SRF for all the candidate genes and there are 40 transcription factors that can bind to promoters of at least 3 of the candidate genes (Fig. 5). This analysis further strengthens the hypothesis that the candidate genes are functionally related and could be regulated by common mechanism. We performed preliminary epigenetic studies to gain information about gene regulation by DNA methylation (5-Azacytidine treatment) and histone acetylation (Trochostatin-A treatment). These experiments done on eight prostate cell lines indicated that DNA methylation and histone acetylation could regulate S100p and CEACAM6 respectively (Fig 6).

Claims

1 A diagnostic method for the detection of cancer cells isolated from a subject comprising determining the expression of two or more genes selected from the group consisting of: lipocalin 2, carcinoembryonic antigen-related cell adhesion molecule, S100 calcium-binding protein P and optionally small proline rich protein 3, wherein over- expression of said gene is indicative of cancer or a predisposition to cancer in said subject.

2. A method according to claim 1 wherein said method detects expression of two or more genes.

3. A method according to claim 1 or 2 wherein said method detects three or more genes.

4. A method according to claim 3 wherein said method detects over expression of lipocalin 2, carcinoembryonic antigen-related cell adhesion molecule and S100 calcium- binding protein P.

5. A method according to any of claims 1 -4 wherein said method comprises:

i) providing an isolated biological sample to be tested; ii) forming a preparation comprising said sample and an oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 7a, 8a, 9a and optionally Figure 10a; a thermostable DNA polymerase, deoxynucleotide triphosphates and co-factors;

iv) analysing the amplified products of said polymerase chain reaction for the presence or absence of a nucleic acid molecule comprising a nucleotide sequence derived from Figure 7a, 8a, 9a and optionally Figure 10a; and optionally

v) comparing the amplified product with a normal matched control.

6. A method according to any of claims 1 -5 wherein said method comprises:

i) providing an isolated biological sample to be tested;

ii) forming a preparation comprising said sample and an antibody or antibodies that specifically binds one or more polypeptide^] in said sample as represented by the amino acid sequences presented in Figures 7b, 8b, 9b or optionally 10b to form an antibody/polypeptide complex; iii) detecting the complex or complexes so formed; and iv) comparing the expression of said polypeptide^] with a normal matched control.

7. A composition comprising two or more antisense RNA molecules wherein said antisense RNA molecule comprise a nucleotide sequence adapted to anneal to a sense nucleotide sequence derived from at least one gene represented by the sense sequence presented in Figure 7a, 8a, 9a, or 10a.

8. A composition according to claim 7 wherein said composition is a pharmaceutical composition.

9. A composition according to claim 7 wherein said composition consists essentially of one or more antisense RNA molecules and physiologically compatible excipients and/or adjuvants.

10. A composition according to any of claims 7-9 wherein said antisense RNA molecule is derived from two of the three nucleotide sequences presented in Figures 7a,

8a, 9a or 10a.

1 1 . A composition according to claim 10 wherein said antisense RNA molecules are derived from each of the nucleotide sequences presented in Figures 7a, 8a and 9a.

12. A composition according to any of claims 7-1 1 wherein said antisense RNA molecule is part of a siRNA or shRNA molecule.

13. A composition according to any of claims 7-12 wherein said antisense RNA molecule is between 19 nucleotides [nt] and 29nt in length.

14. A composition according to claim 13 wherein said antisense RNA consists of 21 nt.

15. A composition according to any of claims 12-14 wherein said antisense, siRNA or shRNA includes modified nucleotides.

16. A composition according to any of claims 8-15 wherein said pharmaceutical composition includes a carrier adapted to deliver said antisense RNA to a cell or tissue.

17. A composition according to any of claims 12-16 wherein said shRNA is part of an expression vector adapted for eukaryotic expression.

18. A composition comprising one or more expression vectors wherein said vector[s] are adapted for expression of at least two nucleic acid molecules comprising nucleotide sequences selected from the group consisting of: i) nucleotide sequences represented by the sequences in Figures 7a, 8a, 9a, or 10a;

19. A composition according to claim 18 wherein said vector[s] are adapted for expression of each nucleic acid molecule comprising the nucleotide sequence represented in Figure 7a, 8a and 9a.

20. A composition according to claim 18 or 19 wherein said adaptation is the provision of a regulatable promoter,

21 . A composition according to claim 20 wherein said promoter is cancer specific.

22. An isolated mammalian cell modified by transfection with a vector composition according to the invention wherein said cell expresses at least two nucleic acid molecules selected from the group consisting of:

i) nucleotide sequences represented by the sequences in Figures 7a, 8a, 9a, or 10a;

23. An isolated cell according to claim 22 wherein said cell over-expresses a nucleic acid comprising a nucleotide sequence as represented in Figure 7a, 8a and 9a.

24. An isolated cell according to claim 22 wherein said cell over-expresses a nucleic acid comprising a nucleotide sequence as represented in Figure 7a, 8a, 9a and 10a.

25. An isolated mammalian cell modified by transfection with an antisense composition according to claim 7 wherein the expression of at least two genes represented by the nucleotide sequences in Figures 7a, 8a, 9a or 10a are down- regulated or ablated.

26. An isolated cell according to claim 25 wherein said cell is transfected with an antisense composition comprising antisense molecules designed with reference to nucleotide sequences represented in Figure 7a, 8a and 9a.

27. An isolated cell according to any of claims 22-26 wherein said mammalian is a human cell.

28. An antisense composition according to any of claims 7-17 for use in the treatment of cancer.

29. A vector composition according to any of claims 18-21 for use in the treatment of cancer.

30. A diagnostic method for the detection of cancer cells isolated from a subject comprising determining the expression of one or more genes selected from the group represented in table 1 wherein over-expression of said gene is indicative of cancer or a predisposition to cancer in said subject.

31 . A composition comprising one or more antisense RNA molecules wherein said antisense RNA molecule comprise a nucleotide sequence adapted to anneal to a sense nucleotide sequence derived from at least one gene represented in table 1 .

32. An isolated mammalian cell modified by transfection with a vector composition according to the invention wherein said cell expresses at least two nucleic acid molecules selected from the nucleotide sequences represented by the genes presented in table 1 .

33. An isolated mammalian cell modified by transfection with an antisense composition according to the invention wherein the expression of at least two genes represented by the nucleotide sequences represented by the genes in table 1 are down- regulated or ablated.