Patent Application
20120282167
The present invention relates to the field of medicine, in particular of oncology. It provides a new method for diagnosing an aggressive tumor and for predicting the sensitivity of a tumor to an epigenetic treatment.
Bladder cancer is the fifth cancer in term of incidence. It can appear as superficial lesions restricted to the urothelium (Ta and carcinoma in situ (CIS)) or to the lamina propria (T1) or as muscle invasive lesions (T2-T4). Two different pathways of tumour progression have been so far described in bladder cancer, the Ta pathway and the CIS pathway. Ta tumours which constitute 50% of bladder tumours at first presentation are superficial papillary tumour usually of low grade which do not invade the basal membrane. Carcinoma-in-situ (CIS) are also superficial tumour which do not invade the basal membrane but are always of high grade.
Ta tumours, despite chirurgical resection associated or not with BCG (Bacillus Calmette-Guerin) therapy, often recur but rarely progress to muscle invasive disease (T2-T4), whereas CIS often progress to T2-T4 tumors. Concerning muscle invasive bladder carcinomas, the standard treatment is cystectomy associated with chemotherapy and/or radiotherapy. Despite this radical treatment, muscle invasive bladder carcinoma remains a deadly disease for most patients.
Accordingly, there is a strong need for an appropriate treatment for bladder tumor of the CIS pathway, in particular for more effective therapeutic protocols.
Moreover, considering that most of anticancer treatments not only cause severe side effects but also are generally physically exhausting for patients and often associated with high costs, the choice of the appropriate therapeutic protocols is of capital importance.
Consequently, practitioners need methods for predicting the sensitivity of a tumor to a particular treatment prior to the actual onset of said treatment.
In a cancer cell, genetic and epigenetic lesions contribute to transcriptional deregulation. Genetic alterations associated with cancer, such as gene mutation, gene amplification, loss of heterozygosity or deletion, may affect single gene or extent to a whole region. Epigenetic changes include alteration of the genomic DNA methylation and histone modification profile. Until recently, epigenetic silencing in cancer has always been envisaged as a local event silencing discrete genes. However, recent findings indicate that large regions of chromosomes can be co-ordinately suppressed, with similar implication as loss of heterozygosity. This phenomenon has been named as long-range epigenetic silencing (LRES).
The mechanism of gene silencing within these regions may be due to DNA and histone modification or histone modification with no associated DNA methylation.
DNA methylation in mammals occurs mainly at cytosine residues in CpG dinucleotide pairs. Short stretches of CpG-dense DNA, known as CpG islands, are typically found associated with gene promoters. Most CpG island promoters are unmethylated, a state associated with active gene transcription. In contrast, CpG island promoters can become de novo methylated in a cancer cell and this methylation is associated with gene silencing.
Histones, in particular H3 and H4, have long tails protruding from the nucleosome which can be covalently modified. Well-described histone modifications include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination, and ADP-ribosylation. Combinations of histone modifications result in different chromatine states and constitute a code, the so-called “histone code”. Typically, acetylation of histone tails is associated with active gene transcription whereas deacetylation is associated with silent gene. Methylation of lysine residues in histone H3 can have opposite effects, e.g. trimethylation of lysine 9 or 27 is associated with silent gene (Barski et al., 2007) whereas trimethylation of lysine 4 is associated with active gene transcription (Koch et al., 2007).
Long-range epigenetic silencing has been described in colon (Frigola et al., 2006) and breast cancers (Novak et al., 2006). These regions have been identified by detecting concordant methylation of adjacent CpG island gene promoters, followed by an examination of histone methylation.
The inventors have herein demonstrated the existence of a particular regional epigenetic silencing (RES) phenotype which is present in tumors belonging to the more aggressive of the two pathways of bladder tumor progression, the carcinoma in situ pathway. Furthermore, the inventors have shown that tumors with this RES phenotype are particularly sensitive to epigenetic therapy.
Accordingly, the present invention concerns a method for determining the RES phenotype of a tumor, wherein the method comprises determining the expression level of at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2, and wherein the over-expression of said genes is indicative of the RES phenotype of the tumor. Optionally, the method further comprises determining the expression level of at least 3 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the absence of over-expression of said genes is indicative of the RES phenotype of the tumor or confirms its RES phenotype. Preferably, the method comprises determining the expression level of a first set of at least 24 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2, and a second set of at least 3 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the over-expression of the genes of the first set and the absence of over-expression of the genes of the second set is indicative of the RES phenotype of the tumor.
Alternatively, the present invention concerns a method for determining the RES phenotype of a tumor, wherein the method comprises determining the number of genes selected from the group consisting of EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9 which are over-expressed and/or determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced, and optionally assessing the expression level of the EZH2 histone methyltransferase in said tumor, and wherein the RES phenotype is defined either by the presence of at least three of said over-expressed genes and/or by the presence of at least three of said silenced regions, and/or by the presence of at least two of said silenced regions and an overexpression of the EZH2 histone methyltransferase. Preferably, the tumor is a bladder tumor.
In an embodiment, the method comprises determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced in said tumor, and the RES phenotype is defined by the presence of at least three of said silenced regions.
In another embodiment, the method comprises determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced, and assessing the expression level of the EZH2 histone methyltransferase in said tumor, and the RES phenotype is defined by the presence of at least two of said silenced regions and an overexpression of the EZH2 histone methyltransferase.
In a further embodiment, the method comprises determining the number of genes selected from the group consisting of EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9 which are over-expressed, and the RES phenotype is defined by the presence of at least three of said over-expressed genes.
In a second aspect, the present invention concerns a method for diagnosing an aggressive tumor, wherein the method comprises determining the RES phenotype in a tumor with the method according to the invention, and wherein the presence of the RES phenotype in said tumor is indicative of an aggressive tumor. Preferably, the tumor is a bladder tumor. In an embodiment, the tumor belongs to the CIS pathway. In another embodiment, the tumor is a muscle-invasive or high grade tumor.
In a third aspect, the present invention concerns a method for predicting the sensitivity of a tumor to an epigenetic therapy, wherein the method comprises determining the RES phenotype in said tumor with the method according to the invention, and wherein the presence of the RES phenotype in said tumor is predictive that said tumor is sensitive to an epigenetic therapy. Preferably, the tumor is a bladder tumor.
In a further aspect, the present invention concerns a method for selecting a patient affected with a tumor for an epigenetic therapy or determining whether a patient affected with a tumor is susceptible to benefit from an epigenetic therapy, wherein the method comprises determining the RES phenotype of said tumor with the method according to the invention, and wherein the presence of the RES phenotype in said tumor is predictive that an epigenetic therapy is indicated for said patient. Preferably, the tumor is a bladder tumor.
In an embodiment, the epigenetic therapy comprises at least one compound selected from the group consisting of histone deacetylase inhibitors, histone methyltransferase inhibitors and histone demethylases, and any combination thereof. Preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2.
In another embodiment, the epigenetic therapy further comprises at least one DNA methyltransferase inhibitor.
In another aspect, the present invention concerns an epigenetic compound for use in the treatment of cancer in a patient affected with a tumor with a RES phenotype. In an embodiment, the epigenetic compound is selected from the group consisting of histone deacetylase inhibitor, histone methyltransferase inhibitor and histone demethylase, and any combination thereof. In another embodiment, the epigenetic compound is used in combination with a DNA methyltransferase inhibitor. Preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2. In a further embodiment, the epigenetic compound is used in combination with another antineoplastic agent.
In a last aspect, the present invention concerns a kit for determining the RES phenotype of a tumor, wherein the kit comprises detection means selected from the group consisting of a pair of primers, a probe and an antibody specific to a) at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2; or to b) the genes EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9; or a DNA chip for determining the RES phenotype of a tumor, wherein the DNA chip comprises a solid support which carries nucleic acids that are specific to a) at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2; or to b) the genes EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9.
A large scale bioinformatics analysis combining paired transcriptome and comparative genomic hybridization (CGH) array data was used to identify regions of neighbouring genes with correlated expression patterns that were not dependent upon changes in copy number. When applied to a series of bladder cancers, this approach led to identify 28 regions of correlated expression that were recognized as candidate regions controlled by epigenetic mechanisms (Stransky et al. 2006).
The inventors have herein demonstrated that some of these regions are silenced by epigenetic alterations involving histone modifications with very rare CpG promoter DNA methylation. They have further showed the existence of a regional epigenetic silencing (RES) phenotype as these particular silenced regions are simultaneously silenced in the same subset of tumors. Strikingly, this subset of tumors belongs to the more aggressive of the two pathways of bladder tumor progression, the carcinoma in situ pathway. Furthermore, in studies herein described, inventor's data reveal that tumors with this RES phenotype are particularly sensitive to epigenetic therapy.
The term “epigenetic compound” as used herein refers to a compound that is able to reverse epigenetic aberrations. An epigenetic compound may be a histone deacetylase inhibitor, a histone methyltransferase inhibitor, a histone demethylase or a DNA methyltransferase inhibitor. Preferably, the epigenetic compound is a histone deacetylase inhibitor, a histone methyltransferase inhibitor or a histone demethylase. More preferably, the epigenetic compound is a histone deacetylase inhibitor and/or a histone methyltransferase inhibitor.
The term “histone deacetylase inhibitor” refers to a compound that interferes with the function of at least one histone deacetylase. A histone deacetylase is a protein that catalyzes removal of an acetyl group from the epsilon-amino group of lysine side chains in histones (H2A, H2B, H3 or H4), thereby reconstituting a positive charge on the lysine side chain and leading to the formation of a condensed and transcriptionally silenced chromatin. In an embodiment, the histone deacetylase inhibitor is selected from the group consisting of a peptide, an antibody, an antigen binding fragment of an antibody, a nucleic acid, an aliphatic acid, a hydroxamic acid, a benzamide, depudecin, and an electrophilic ketone, and a combination thereof. In a particular embodiment, the histone deacetylase inhibitor is an oligonucleotide that inhibits expression or function of histone deacetylase, such as an antisense molecule or a ribozyme. Alternatively, the histone deacetylase inhibitor is a dominant negative fragment or variant of histone deacetylase. Examples of histone deacetylase inhibitors include, but are not limited to, trichostatin A, vorinostat (suberoylanilide hydroxamic acid or SAHA), valproic acid, belinostat (PXD101), Panobinostat (LBH-589), MS-275, N-acetyldinaline (CI-994), depudecin, oxamflatin, bishydroxyamic acid, MGCD0103, Scriptaid, apicidin, derivatives of apicidin, benzamide, derivatives of benzamide, FR901228, FK228, trapoxin A, trapoxin B, HC-toxin, chlamydocin, Cly-2, WF-3161, Tan-1746, pyroxamide, NVP-LAQ824, butyrate, phenylbutyrate, hydroxyamic acid derivatives, cyclic hydroxamic acid-containing peptide (CHAP), m-carboxycinnamic acid bishydroxamic acid (CBHA), suberic bishydroxyamic acid and azelaic bishydroxyamic acid, and a salt thereof. In a particular embodiment, the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, vorinostat, valproic acid, panobinostat and belinostat. In a preferred embodiment, the histone deacetylase inhibitor is vorinostat. More preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2. Still more preferably, the compound has specificity for the HDAC of class I, in particular for the HDAC1, HDAC2 and/or HDAC3, preferably HDAC1 and/or HDAC2. In particular, the inhibitor may be MS-275 or SK-7041, SK-7068, Pyroxamide, Apicidin, Depsipeptides, MGCD-0103, Depudecin.
The term “histone methyltransferase inhibitor” refers to a compound that interferes with the function of at least one histone methyltransferase. A histone methyltransferase is a histone-lysine N-methyltransferase (registry number EC 2.1.1.43) or a histone-arginine N-methyltransferase (registry number EC 2.1.1.23). These enzymes catalyze the transfer of one to three methyl groups from the cofactor S-Adenosyl methionine to lysine or arginine residues of histone proteins. In an embodiment, the histone methyltransferase inhibitor is selected from the group consisting of a peptide, an antibody, an antigen binding fragment of an antibody, a nucleic acid and a drug, and a combination thereof. In a particular embodiment, the histone methyltransferase inhibitor is an oligonucleotide that inhibits expression or function of histone methyltransferase, such as an antisense molecule or a ribozyme. Alternatively, the histone methyltransferase inhibitor is a dominant negative fragment or variant of histone methyltransferase. In a particular embodiment, the histone methyltransferase inhibitor inhibits a histone methyltransferase selected from the group consisting of EZH2, G9A, ESET, SUV39h1, SUV39h2 and Eu-HMTase1. In a particular embodiment, the histone methyltransferase inhibitor is selected from the group consisting of BIX-01294 (Kubicek et al., 2007), Chaetocin (Greiner et al., 2005) and 3-Deazaneplanocin A. In another particular embodiment, the histone methyltransferase inhibitor is a siRNA which specifically inhibits the expression of EZH2.
The term “histone demethylase” refers to proteins which are able to reverse histone methylation. Examples of histone demethylases include JMJD2 family of proteins (Whetstine et al., 2006), in particular JMJD2C, JMJD3, JMJD1A, JHDM3 family and JMJD3/UTX proteins. In particular, proteins of the JHDM1 family include JHDM1A, proteins of the JHDM3/JMJD2 subfamily include JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1 and JMJD2D, proteins of the JARID subfamily include JARID1A, JARID B, JARID C and JARID D, proteins of the UTX/UTY sub-family include UTX and JMJD3, proteins of the JHDM2 subfamily include JHDM2A, JHDM2B and JHDM2C. The histone demethylase may further include the peptidyl arginine deiminase PADI4 or the flavin-dependent amine oxidase LSD1. In a preferred embodiment, the histone demethylase is able to reverse H3K9me3 and/or H3K27me3 histone modification.
The common nomenclature of histone modifications is as follows: first, the name of the histone (e.g H3), second the single letter amino acid abbreviation (e.g. K for Lysine) and the amino acid position in the protein, and third the type of modification (Me: methyl, P: phosphate, Ac: acetyl, Ub: ubiquitin). As example, H3K9me3 denotes the trimethylation of the 9th residue (a lysine) from the N-terminal of the H3 protein and H3K9ac denotes the acetylation of the 9th residue (a lysine) from the N-terminal of the H3 protein.
The term “DNA methyltransferase inhibitor” refers to a compound that interferes with the function of at least one DNA methyltransferase. A DNA methyltransferase (DNMT) is an enzyme that catalyzes the transfer of a methyl group to DNA. Four active DNA methyltransferases have been identified in mammals, namely DNMT1, DNMT2, DNMT3A and DNMT3B. The DNA methyltransferase inhibitor may be selected from the group consisting of a peptide, an antibody, an antigen binding fragment of an antibody, a nucleic acid and a drug, and a combination thereof. In a particular embodiment, the DNA methyltransferase inhibitor is an oligonucleotide that inhibits expression or function of DNA methyltransferase, such as an antisense molecule or a ribozyme. Alternatively, the DNA methyltransferase inhibitor is a dominant negative fragment or variant of DNA methyltransferase. Examples of DNA methyltransferase inhibitors include, but are not limited to, 5-azacytidine (5-azaCR), decitabine (5-aza-2′-deoxycytidine or 5-aza-CdR), 5-fluoro-2′-deoxycytidine, 5,6-dihydro-5-azacytidine, procaine, (−)-epigallocatechin-3-gallate (EGCG), zebularine (1-(beta-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one), NSC 303530 (Siedlecki et al., J Med. Chem. 2006, 49(2):678-83), NSC 401077 (RG108), procainamide, hydralazine, psammaplin A and MG98. Other examples include compounds described in patent applications WO 2008/033744, WO 99/12027, WO 2005/085196, EP 1 844 062 and WO 2006/060382, and in the article of Siedlecki et al. (Siedlecki et al., 2006).
The term “epigenetic therapy” as used herein refers to a treatment involving at least one epigenetic compound. In an embodiment, an “epigenetic treatment” or “epigenetic therapy” refers to a treatment involving at least a histone deacetylase inhibitor, a histone methyltransferase inhibitor and/or a histone demethylase, preferably involving at least a histone deacetylase inhibitor. In a preferred embodiment, an epigenetic treatment refers to a treatment involving at least one histone deacetylase inhibitor and at least one histone methyltransferase inhibitor. In a particular embodiment, an epigenetic treatment refers to a treatment involving at least a histone deacetylase inhibitor, a histone methyltransferase inhibitor and/or a histone demethylase, in combination with a DNA methyltransferase inhibitor.
The term “cancer” or “tumor” as used herein refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases). Typical cancers are breast, stomach, oesophageal, sarcoma, ovarian, endometrium, bladder, cervix uteri, rectum, colon, lung or ORL cancer, paediatric tumours (neuroblastoma, glyoblastoma multiforme), lymphoma, leukaemia, myeloma, seminoma, Hodgkin and malignant hemopathies. Preferably, the cancer is a solid cancer. More preferably, the cancer is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Even more preferably, the cancer is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. Even more preferably, the cancer is a bladder cancer. In a particular embodiment, the cancer is an epithelial-derived cancer.
Based on the microscopic appearance of cancer cells, pathologists commonly describe tumor grade by four degrees of severity: Grades 1, 2, 3, and 4. The cells of Grade 1 tumors resemble normal cells, and tend to grow and multiply slowly. Conversely, the cells of Grade 3 or Grade 4 tumors do not look like normal cells of the same type. Grade 3 and 4 tumors tend to grow rapidly and spread faster than tumors with a lower grade. Usually, tumors are grading as follow: G1: Well-differentiated (Low grade); G2: Moderately differentiated (Intermediate grade); G3: Poorly differentiated (High grade); and G4: Undifferentiated (High grade). As used herein, a high grade tumor is a tumor of G3 or G4 grade.
By “bladder tumor” is intended herein urinary bladder tumor, bladder cancer, bladder carcinoma or urinary bladder cancer, and bladder neoplasm or urinary bladder neoplasm. A bladder tumor can be a bladder carcinoma or a bladder adenoma. The most common staging system for bladder tumors is the TNM (tumor, node, metastasis) system. This staging system takes into account how deep the tumor has grown into the bladder, whether there is cancer in the lymph nodes and whether the cancer has spread to any other part of the body. The following stages are used to classify the location, size, and spread of the cancer, according to the TNM staging system: Stage 0 (CIS or Ta): Cancer cells are found only on the inner lining of the bladder; Stage I (T1): Cancer cells have started to grow into the connective tissue beneath the bladder lining; Stage II (T2): Cancer cells have grown through the connective tissue into the muscle; Stage III (T3): Cancer cells have grown through the muscle into the fat layer; Stage IV (T4): Cancer cells have proliferated to the lymph nodes, pelvic or abdominal wall, and/or other organs. In an embodiment, the bladder tumor is a bladder carcinoma. In a particular embodiment, the bladder tumor belongs to the carcinoma in situ (CIS) pathway. In another particular embodiment, the bladder tumor is a muscle-invasive tumor, i.e. T2-T4 tumor or a high grade tumor (G3 or G4). As used herein, the term “aggressive bladder tumor” refers to a high-grade (G3 or G4) tumor, T2-T4 tumors and tumors of the CIS pathway. Preferably, the term “aggressive bladder tumor” refers to tumors of the CIS pathway.
As used herein, the term “treatment”, “treat” or “treating” refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease. In particular, the term “to treat a cancer”, “treating a cancer”, “to treat a tumor” or “treating a tumor” means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient.
As used herein, the term “subject” or “patient” refers to an animal, preferably to a mammal, even more preferably to a human, including adult, child and human at the prenatal stage. However, the term “subject” or “patient” can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among others, that are in need of treatment.
The term “sample”, as used herein, means any sample containing cells derived from a subject, preferably a sample which contains nucleic acids. Examples of such samples include fluids such as blood, plasma, saliva, urine and seminal fluid samples as well as biopsies, organs, tissues or cell samples. The sample may be treated prior to its use, e.g. in order to render nucleic acids available. The term “cancer sample” or “tumor sample” refers to any sample containing tumoral cells derived from a patient, preferably a sample which contains nucleic acids. Preferably, the sample contains only tumoral cells. The term “normal sample” refers to any sample which does not contain any tumoral cell.
The methods of the invention as disclosed below, may be in vivo, ex vivo or in vitro methods, preferably in vitro methods.
In a first aspect, the present invention concerns a method for identifying chromosomal regions which could be involved in the RES phenotype of a given type of tumors, said method comprising: (a) identifying chromosomal regions with correlated expression; (b) excluding tumors with copy-number alteration; (c) selecting regions presented downregulation; (d) selecting regions containing at least 3 downregulated or non expressed contiguous genes; and (e) selecting regions silenced by histone modification.
In steps (a) and (b), copy number-independent regions of correlated expression are identified by combining transcriptome and CGH array data for a set of tumors belonging to a type of tumors of interest. For example, the identification of such chromosomal regions has been described for a set of bladder tumors in the article of Stransky et al. (Stransky et al., 2008; the disclosure of which is incorporated herein by reference). In summary, a transcriptome correlation map (TCM) which assesses the correlation which exists between the expression of a gene and those of neighbors is established (step (a)). CGH array analyses of the same set of tumors lead to identification of tumors that show genetic losses or gains. A new TCM is then recalculated, with exclusion of these tumors with copy-number alterations, and chromosomal regions with copy number-independent are identified (step (b)).
In step (c), regions with correlated expression due to down-regulation are selected among regions selected in step (b). For each correlated gene, the ratio between its expression value in each tumor sample and its mean expression in normal samples is calculated. These expression ratios are then used to cluster, for each region, all normal and tumor samples. For selected regions, the deregulation is represented by all or a subset of tumors. Preferably, at least three normal samples are used, more preferably at least five.
In step (d), regions containing a stretch of downregulated or non-expressed genes are selected among regions selected in step (c).
Finally, in step (e), regions silenced by histone modifications are selected among regions selected in step (d). These regions comprise very rare methylated promoter and thus DNA methylation is not significant enough to explain the silencing of these regions.
These regions are identified based on the study of a set of tumors of the same type but of varying grade and stage. Preferably, the set comprises at least 20 tumors. More preferably, the set comprises at least 50 tumors.
This method may be applied on sets of tumors of any type of cancer and chromosomal regions which could be involved in the RES phenotype in said cancer may be thus identified. Based on a set of bladder tumors, the chromosomal regions implicated in the RES phenotype in bladder cancer have been identified. These regions are regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B.
The present invention concerns a method for determining the RES phenotype of a tumor, wherein the method comprises determining the number of genes selected from the group consisting of EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9 which are over-expressed and/or determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced, and optionally assessing the expression level of the EZH2 histone methyltransferase in said tumor, and wherein the RES phenotype is defined either by the presence of at least three of said over-expressed genes and/or by the presence of at least three of said silenced regions, and/or by the presence of at least two of said silenced regions and an overexpression of the EZH2 histone methyltransferase.
In an embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor.
In an embodiment, the method further comprises the step of providing a tumor sample from a subject.
Generally, the expression level of a gene is determined as a relative expression level. More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.
In a particular embodiment, gene expression is determined by measuring the quantity of mRNA. For example the nucleic acid contained in the sample (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Preferably quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). Amplification primers may be easily designed by the skilled person.
In another embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art.
Gene expression in samples may be normalized by using expression levels of proteins which are known to have stable expression such as RPLPO (acidic ribosomal phosphoprotein PO), TBP (TATA box binding protein), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), β-actin or 18rRNA.
Gene expression levels in tumor sample are then compared with gene expression levels in normal sample. Preferably, the normal sample is provided from the same tissue type than the tumor sample. In an embodiment, the tumor sample is a sample of bladder tumor and the normal sample is a sample of normal urothelium. The normal sample may be obtained from the subject affected with the cancer or from another subject, preferably a normal or healthy subject, i.e. a subject who does not suffer from a cancer.
A gene is considered as silenced in tumor sample if, after normalization, the expression level of this gene is at least 1.5-fold lower than its expression level in the normal sample. Preferably, a gene is considered as silenced in tumor sample if, after normalization, the expression level of this gene is at least 2, 3, 4 or 5-fold lower than its expression level in the normal sample.
A gene is considered as over-expressed in tumor sample if, after normalization, the expression level of this gene is at least 1.5-fold higher than its expression level in the normal sample. Preferably, a gene is considered as over-expressed in tumor sample if, after normalization, the expression level of this gene is at least 2, 3, 4, or 5-fold higher than its expression level in the normal sample. In a preferred embodiment, a gene is considered as over-expressed in a tumor sample if, after normalization, the expression level of this gene is at least 2-fold higher than its expression level in the normal sample.
In an embodiment, the method for determining the RES phenotype of a tumor comprises determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced in said tumor, wherein the RES phenotype is defined by the presence of at least three of said silenced regions.
Chromosomal regions are identified according to the International System for Human Cytogenetic Nomenclature (ISCN) fixed by the Standing Committee on Human Cytogenetic Nomenclature. Short arm locations are labeled p and long arms q. Each chromosome arm is divided into regions labeled p1, p2, p3 etc., and q1, q2, q3, etc., counting outwards from the centromere. Regions are delimited by specific landmarks, which are consistent and distinct morphological features, such as the ends of the chromosome arms, the centromere and certain bands. Regions are divided into bands labeled p11, p12, p13, etc., sub-bands labeled p11.1, p11.2, etc., and sub-sub-bands e.g. p11.21, p11.22, etc., in each case counting outwards from the centromere.
The region 2-7 is considered as silenced if at least three contiguous genes comprised in this region and selected from the group consisting of HOXD4, HOXD3, HOXD1 and MTX2 genes are silenced. These genes are located on chromosome 2 in location 2q31. In an embodiment, HOXD4, HOXD3 and HOXD1 are silenced. In another embodiment, HOXD3, HOXD1 and MTX2 are silenced. In a preferred embodiment, HOXD4, HOXD3, HOXD1 and MTX2 are silenced.
The region 3-2 is considered as silenced if at least three contiguous genes comprised in this region and selected from the group consisting of VILL, PLCD1, DLEC1 and ACAA1 genes are silenced. These genes are located on chromosome 3 in location 3p22-p21.3. In an embodiment, VILL, PLCD1 and DLEC1 are silenced. In another embodiment, PLCD1, DLEC1 and ACAA1 are silenced. In a preferred embodiment, VILL, PLCD1, DLEC1 and ACAA1 are silenced.
The region 3-5 is considered as silenced if at least three contiguous genes comprised in this region and selected from the group consisting of TCTA, AMT, NICN1, DAG1, BSN, APEH, RNF123 and GMPPB genes are silenced. These genes are located on chromosome 3 in location 3p21-24.3. In an embodiment, TCTA, AMT and NICN1 are silenced. In another embodiment, AMT, NICN1 and DA G are silenced. In another embodiment, NICN1, DA G and BSN are silenced. In a further embodiment, DAG1, BSN and APEH are silenced. In another embodiment, BSN, APEH and RNF123 are silenced. In a further embodiment, APEH, RNF123 and GMPPB are silenced. In a preferred embodiment, TCTA, AMT, NICN1, DAG1, BSN, APEH, RNF123 and GMPPB are silenced.
The region 7-2 is considered as silenced if at least three contiguous genes comprised in this region and selected from the group consisting of SKAP2, HOXA1, HOXA2, HOXA3, HOXA4 and HOXA5 genes are silenced. These genes are located on chromosome 7 in location 7p15. In an embodiment, SKAP2, HOXA1 and HOXA2 are silenced. In another embodiment, HOXA1, HOXA2 and HOXA3 are silenced. In another embodiment, HOXA2, HOXA3 and HOXA4 are silenced. In a further embodiment, HOXA3, HOXA4 and HOXA5 are silenced. In a preferred embodiment, SKAP2, HOXA1, HOXA2, HOXA3, HOXA4 and HOXA5 are silenced.
The region 14-1 is considered as silenced if at least three contiguous genes comprised in this region and selected from the group consisting of CMTM5, MYH6, MYH7, THTPA, AP1G2, DHRS2 and DHRS4 genes are silenced. These genes are located on chromosome 14 in location 14q1-12. In an embodiment, CMTM5, MYH6 and MYH7 are silenced. In another embodiment, THTPA, AP1G2 and DHRS2 are silenced. In a further embodiment, AP1G2, DHRS2 and DHRS4 are silenced. In a preferred embodiment, CMTM5, MYH6, MYH7, THTPA, AP1G2, DHRS2 and DHRS4 are silenced.
The region 19-3A is considered as silenced if at least three contiguous genes comprised in this region and selected from the group consisting of CYP4F3, CYP4F12, CYP4F2 and CYP4F11 genes are silenced. These genes are located on chromosome 19 in location 19p13. In an embodiment, CYP4F3, CYP4F12 and CYP4F2 are silenced. In another embodiment, CYP4F12, CYP4F2 and CYP4F11 are silenced. In a preferred embodiment, CYP4F3, CYP4F12, CYP4F2 and CYP4F11 are silenced.
The region 19-3B is considered as silenced if at least B3GNT3, INSL3 and JAK3 genes comprised in this region are silenced. These genes are located on chromosome 19 in location 19p13.
In an embodiment, the RES phenotype is defined by the presence of at least 3 of the silenced chromosomal regions described above. In another embodiment, the RES phenotype is defined by the presence of at least 4 of said regions. In a further embodiment, the RES phenotype is defined by the presence of at least 5 of said regions.
In another embodiment, the method for determining the RES phenotype of a tumor comprises determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced, and assessing the expression level of the EZH2 histone methyltransferase in said tumor, wherein the RES phenotype is defined by the presence of at least two of said silenced regions and an overexpression of the EZH2 histone methyltransferase.
The number of chromosomal regions selected from the group consisting of regions 2-7,3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced, may be assessed as described above.
EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that targets lysine-27 of histone H3. The expression of this enzyme may be assessed by any method known by the skilled person such as quantitative or semi quantitative RT-PCR as well as real-time quantitative or semi quantitative RT-PCR, as described above.
In a particular embodiment, the RES phenotype is defined by the presence of at least three of silenced chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B and an overexpression of the EZH2 histone methyltransferase.
In a further embodiment, the method for determining the RES phenotype of a tumor comprises determining the expression level of at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2, and wherein the over-expression of said genes is indicative of the RES phenotype of the tumor. Optionally, the method comprises determining the expression level of at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C50orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8 and SULF2, and wherein the over-expression of said genes is indicative of the RES phenotype of the tumor. Alternatively, the method comprises determining the expression level of at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, and AEBP1, and wherein the over-expression of said genes is indicative of the RES phenotype of the tumor. Preferably, the method comprises determining the expression level of at least 25, 30, 35 or 40 genes selected in the above-mentioned lists. The method may comprise determining the expression level of 20, 25, 30, 35 or 40 genes selected in the above-mentioned lists. In a particular embodiment, the method comprises determining the expression level of the genes of the above-mentioned lists. In a particular aspect, the genes are selected according to the order of the list. For instance, the 20 genes may be the followings: LC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, and IFI30. The 24 genes may be the followings: SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, and AEBP1. The 25 genes may be the followings: SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, and GBP5. The 30 genes may be the followings: SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD and CIS. The 35 genes may be the followings: SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C50orf13 and DSC2. Alternatively, the genes may also be selected randomly in the list.
In addition and in the context of this embodiment, the method may further comprises determining the expression level of at least 3, 5 or 7 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the absence of over-expression of said genes is indicative of the RES phenotype of the tumor or confirms its RES phenotype. Alternatively, the method may further comprise the expression level of at least 3, 5 or 7 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the over-expression of said genes is indicative of the absence of the RES phenotype of the tumor or refutes its RES phenotype. In case discrepancy between RES+ and RES− markers, the RES status of the tumor may be determined by another method disclosed herein, preferably by the method based on the measurement of the chromosomal regions silencing. Optionally, the group may consist of the genes IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1 and HS6ST3. The method may comprise determining the expression level of 3, 5, 7 or 9 genes selected in the above-mentioned lists. In a particular aspect, the genes are selected according to the order of the list. For instance, the 3 genes may be the followings: ANXA10, IGF2 and B3GALNT1. The 5 genes may be the followings: ANXA10, IGF2, B3GALNT1, EPHB6 and SEMA6A. The 7 genes may be the followings: ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57 and SLC15A1. Alternatively, the genes may also be selected randomly in the list.
Alternatively, the method for determining the RES phenotype of a tumor comprises determining the expression level of a first set of at least 20 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2, and a second set of at least 3 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the over-expression of the genes of the first set and the absence of over-expression of the genes of the second set is indicative of the RES phenotype of the tumor. Preferably, the method comprises determining the expression level of at least 25, 30, or 40 genes selected in the above-mentioned lists for the first set and of at least 5 or 7 genes selected in the above-mentioned lists for the second set. The method may comprise determining the expression level of 20, 25, 30, 35 or 40 genes selected in the above-mentioned lists for the first set and of 3, 5, 7 or 9 genes selected in the above-mentioned lists for the second set. In a particular embodiment, the method comprises determining the expression level of the genes of the above-mentioned lists. Alternatively, the genes may also be selected randomly in the list. Optionally, the method comprises determining the expression level of a first set of at least 24 genes selected from the group consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2, AEBP1, GBP5, MSN, BGN, CTHRC1, MMD, C1S, IGK@, COL5A2, THY1, C5orf13, DSC2, SFRP2, NID2, TIMP2, ADAMTS12, GPX8, SULF2, and a second set of at least 3 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the over-expression of the genes of the first set and the absence of over-expression of the genes of the second set is indicative of the RES phenotype of the tumor. More preferably, the method comprises determining the expression level of a first set of at least 24 genes consisting of SLC16A1, SULF1, POSTN, LOX, FN1, CHI3L1, SFRP4, TNC, COL3A1, FAP, CXCL10, PLA2G7, GREM1, COL1A2, COL1A1, GUCY1A3, PFTK1, COL6A3, FBN1, IFI30, CXCL9, PRRX1, AHNAK2 and AEBP1, and a second set of at least 3 genes selected from the group consisting of ANXA10, IGF2 and B3GALNT1, and wherein the over-expression of the genes of the first set and the absence of over-expression of the genes of the second set is indicative of the RES phenotype of the tumor.
Finally, the method for determining the RES phenotype of a tumor comprises determining the expression level of at least 3, 5 or 7 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20, and wherein the over-expression of said genes is indicative of the absence of the RES phenotype of the tumor. Optionally, the group may consist of IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1 and HS6ST3. The method may comprise determining the expression level of 3, 5, 7 or 9 genes selected in the above-mentioned lists. In a particular aspect, the genes are selected according to the order of the list. Alternatively, the genes may also be selected randomly in the list.
The expression level of a gene is determined as detailed above.
In another embodiment, the method for determining the RES phenotype of a tumor comprises determining the number of genes selected from the group consisting of EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9 which are over-expressed, wherein the RES phenotype is defined by the presence of at least three of said over-expressed genes. Preferably, genes are selected from the group consisting of EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161 and CSRP2.
The Gene ID numbers and Gene Names for the genes disclosed herein are the following:
The expression of these genes may be assessed by any method known by the skilled person such as quantitative or semi quantitative RT-PCR as well as real-time quantitative or semi quantitative RT-PCR, as described above.
In a particular embodiment, the RES phenotype is defined by the presence of at least four of said over-expressed genes.
In further embodiment, the method for determining the RES phenotype of a tumor comprises determining the number of chromosomal regions selected from the group consisting of regions 2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B which are silenced and determining the number of genes selected from the group consisting of EZH2, CDC25B, TUBB3, CDH2, CXCL3, CXCL6, MLLT11, CXCL2, CTSL2, NFIL3, GPR161, CSRP2 and HDAC9 which are over-expressed, wherein the RES phenotype is defined by the presence of at least two of said silenced regions and the presence of at least three of said over-expressed genes.
The number of silenced chromosomal regions and the number of over-expressed genes are determined as described above.
In a particular embodiment, the RES phenotype is defined by the presence of at least three of said silenced regions and the presence of at least three of said over-expressed genes.
The present invention also concerns a method for diagnosing an aggressive tumor in a subject, wherein the method comprises determining the RES phenotype in a tumor with the method according to the invention, as described above, and wherein the presence of the RES phenotype in said tumor is indicative of an aggressive tumor.
The presence of the RES phenotype in a tumor may be determined by the method of the invention as described above.
In an embodiment, the method further comprises the step of providing a sample from a subject affected with a cancer or suspected to be affected with a cancer.
In a particular embodiment, the aggressive tumor belongs to the CIS pathway.
In another embodiment, the aggressive tumor is a muscle-invasive or high grade tumor.
In a preferred embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor.
The present invention also concerns a method for providing useful information for the diagnosis of an aggressive tumor in a subject, wherein the method comprises determining the RES phenotype in a tumor with the method according to the invention, as described above, and wherein the presence of the RES phenotype in a tumor is indicative of an aggressive tumor. In an embodiment, the method further comprises the step of providing a sample from the subject. In a preferred embodiment, the tumor is a bladder tumor.
The inventors have herein shown that tumors with RES phenotype belong to aggressive subset of tumors. Accordingly, the present invention concerns a method for predicting or monitoring clinical outcome of a subject affected with a tumor, wherein the method comprises determining the RES phenotype in a tumor with the method according to the invention, as described above, and wherein the presence of the RES phenotype in a tumor is indicative of a poor prognosis.
In an embodiment, the method further comprises the step of providing a cancer sample from the subject.
In a particular embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor.
The term “poor prognosis”, as used herein, refers to an early disease progression and a decreased patient survival and/or an increased metastasis formation. This prognosis is usually associated with aggressive tumors which are frequently of high grade and progress to muscle-invasive tumors.
The inventors have herein demonstrated that tumors with the RES phenotype are particularly sensitive to epigenetic therapy. Accordingly, the present invention concerns a method for predicting the sensitivity of a tumor to an epigenetic therapy, wherein the method comprises determining the RES phenotype in said tumor with the method according to the invention, as described above, and wherein the presence of the RES phenotype in said tumor is predictive that said tumor is sensitive to an epigenetic therapy.
In an embodiment, the method further comprises the step of providing a cancer sample from the subject.
In a particular embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor.
In a preferred embodiment, the epigenetic therapy comprises at least one compound selected from the group consisting of histone deacetylase inhibitor, histone methyltransferase inhibitor and histone demethylase, and any combination thereof.
Preferably, the epigenetic therapy comprises at least one histone deacetylase inhibitor. More preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2. Still more preferably, the epigenetic therapy comprises at least one histone deacetylase inhibitor and at least one histone methyltransferase inhibitor. In a particular embodiment, the epigenetic therapy comprises a histone deacetylase inhibitor and a histone methyltransferase inhibitor.
In a particular embodiment, the epigenetic therapy further comprises at least one DNA methyltransferase inhibitor.
A tumor is sensitive to an epigenetic therapy if the administration of such therapy induces a decreased growth rate of the tumoral cells and/or an inhibition of the growth of tumoral cells and/or the death of tumoral cells.
The present invention further concerns a method for selecting a patient affected with a tumor for an epigenetic therapy or determining whether a patient affected with a tumor is susceptible to benefit from an epigenetic therapy, wherein the method comprises determining the RES phenotype of said tumor with the method according to the invention, and wherein the presence of the RES phenotype in said tumor is predictive that an epigenetic therapy is indicated for said patient.
In an embodiment, the method further comprises the step of providing a cancer sample from the subject.
In a particular embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor.
In a preferred embodiment, the epigenetic therapy comprises at least one compound selected from the group consisting of histone deacetylase inhibitor, histone methyltransferase inhibitor and histone demethylase, and any combination thereof. Preferably, the epigenetic therapy comprises at least one histone deacetylase inhibitor. More preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2. Still more preferably, the epigenetic therapy comprises at least one histone deacetylase inhibitor and at least one histone methyltransferase inhibitor.
In a particular embodiment, the epigenetic therapy further comprises at least one DNA methyltransferase inhibitor.
The present invention also concerns an epigenetic compound for use in the treatment of cancer in a patient affected with a tumor with a RES phenotype.
The presence of the RES phenotype in a tumor may be assessed by any method of the invention, as described above.
In an embodiment, the epigenetic compound is selected from the group consisting of histone deacetylase inhibitor, histone methyltransferase inhibitor and histone demethylase, and any combination thereof.
In a preferred embodiment, the epigenetic compound is a histone deacetylase inhibitor. Preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2. More preferably, the histone deacetylase inhibitor is used in combination with a histone methyltransferase inhibitor.
In a particular embodiment, the epigenetic compound is used in combination with a DNA methyltransferase inhibitor.
In another particular embodiment, the epigenetic compound is used in combination with an antineoplastic agent.
An “antineoplastic agent” is an agent with anti-cancer activity that inhibits or halts the growth of cancerous cells or immature pre-cancerous cells, kills cancerous cells or immature pre-cancerous cells, increases the susceptibility of cancerous or pre-cancerous cells to other antineoplastic agents, and/or inhibits metastasis of cancerous cells. These agents may include chemical agents as well as biological agents. Examples include, without limitation, 5-aza-2′deoxycytidine, 17-AAG (17-N-Allylamino-17-demethoxygeldanamycin), tretinoin (ATRA), bortezomib, cisplatin, carboplatin, oxaliplatin, paclitaxel, bevacizumab, tamoxifen, leucovorin, docetaxel, transtuzumab, etoposide, flavopiridol, 5-fluorouracil, irinotecan, TRAIL (TNF-related apoptosis-inducing ligand), LY294002, PD184352, perifosine, Bay 11-7082, gemcitabine, bicalutamide, zoledronic acid, cis-retinoic acid, MK-0457, imatinib, desatinib, sorafenib, temozolomide, actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicine, idarubicine, epirubicin, bleomycin, plicamycin and mitomycin. Antineoplastic agents may also include radiotherapeutic agents such as X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiation.
In a particular embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor.
The present invention further concerns a method for treating a cancer in a patient affected with a tumor with a RES phenotype, said method comprising the administration of a therapeutically effective amount of an epigenetic compound to said patient.
The term “therapeutically effective amount” refers to that amount of a therapy which is sufficient to reduce or ameliorate the severity, duration and/or progression of a disease or one or more symptoms thereof. As used herein, this term refers to that amount of an epigenetic compound which is sufficient to destroy, modify, control or remove primary, regional or metastatic cancer tissue, ameliorate cancer or one or more symptoms thereof, or prevent the advancement of cancer, cause regression of cancer, or enhance or improve the therapeutic effect (s) of another therapy (e.g., a therapeutic agent). This term may also refer to the amount of an epigenetic compound sufficient to delay or minimize the spread of cancer or sufficient to provide a therapeutic benefit in the treatment or management of cancer. Further, a therapeutically effective amount with respect to an epigenetic compound means that amount of epigenetic compound alone, or in combination with other therapeutic agent, that provides a therapeutic benefit in the treatment or management of cancer.
In an embodiment, the method further comprises determining the RES phenotype of said tumor with the method of the present invention as described above.
In a particular embodiment, the tumor is selected from the group consisting of bladder cancer, colorectal cancer, oesophageal cancer, neuroblastoma, breast cancer and lung cancer. Preferably, the tumor is selected from the group consisting of bladder cancer, colorectal cancer and breast cancer. More preferably, the tumor is a bladder tumor. In an embodiment, the epigenetic compound is selected from the group consisting of histone deacetylase inhibitor, histone methyltransferase inhibitor and histone demethylase, and any combination thereof. Preferably, the epigenetic compound is a histone deacetylase inhibitor. More preferably, the compound is an inhibitor of histone deacetylases HDAC1, HDAC2 and/or HDAC3, more preferably of HDAC1 and/or HDAC2. Still more preferably, the histone deacetylase inhibitor is administrated simultaneously or sequentially with a histone methyltransferase inhibitor.
The present invention also concerns:
In a particular embodiment, the kit or DNA chip comprises detection means or nucleic acids that are specific to:
Optionally, the kit or DNA chip may further comprise detection means or nucleic acids that are specific to at least 3, 5 or 7 genes selected from the group consisting of ANXA10, IGF2, B3GALNT1, EPHB6, SEMA6A, CXorf57, SLC15A1, HS6ST3 and KRT20. In particular, the kit or DNA chip may further comprise detection means or nucleic acids that are specific to ANXA10, IGF2 and B3GALNT1.
Accordingly, the present invention relates to the kit or DNA chip comprising detection means or nucleic acids that are specific to:
Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labeled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labeled hybridized complexes are then detected and can be quantified or semi-quantified. Labeling may be achieved by various methods, e.g. by using radioactive or fluorescent labeling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, et 2006).
The kit or DNA chip of the invention includes detection means for the genes as defined above in the method for determining the RES phenotype. In a particular aspect, the kit or DNA chip does not include means for detecting more than 100, 80, 70, or 60 genes.
The kit or DNA chip of the invention can further comprise detection means or nucleic acids for control gene, for instance a positive and negative control or a nucleic acid for an ubiquitous gene in order to normalize the results.
All references cited in this specification are incorporated by reference.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.”
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
The following examples are given for purposes of illustration and not by way of limitation.
The analysis of the gene expression profiles and genomic alterations of 57 urothelial bladder carcinomas have been previously reporter (Stransky et al., 2006). These carcinomas were obtained from 53 patients included between 1988 and 2001 in the prospective database established in 1988 at the Department of Urology of Henri Mondor Hospital. The tumor samples came from 16 Ta, 9 T1, 6 T2, 13 T3 and 13 T4 tumors. The flash-frozen tumor samples were stored at −80° C. immediately after transurethral resection or cystectomy. All tumor samples contained more than 80% tumor cells, as assessed by H&E staining of histological sections adjacent to the samples used for transcriptome and genome analyses. Five normal urothelial samples, obtained as described in the article of Diez de Medina et al. (Diez de Medina et al., 1997) were also used for transcriptome analysis. An independent set of 40 human bladder tumors, containing 10 Ta, 6 T1, 6 T2, 7 T3 and 11 T4 tumors, was used to validate the existence of the RES phenotype. These tumors, provided by the Henri Mondor and Foch hospitals and Institut Gustave Roussy, were obtained from 40 patients who underwent surgery between 1993 and 2006. All patients provided informed consent and the study was approved by the ethics committees of the different hospitals.
RNA and DNA were extracted from the samples by cesium chloride density centrifugation (Chirgwin et al., 1979). The concentration and integrity/purity of each RNA sample were determined with the RNA 6000 LabChip Kit (Agilent Technologies) and an Agilent 2100 bioanalyzer. DNA purity was also assessed from the ratio of absorbances at 260 and 280 nm. DNA concentration was determined with a Hoechst dye-based fluorescence assay49. RNA and DNA were extracted from cell lines with Qiagen extraction kits (Qiagen, Courtaboeuf, France).
The bladder cancer cell lines TCCSUP, HT1376, RT112, T24, MGHU3 and CL1207 were cultured in DMEM F-12 Glutamax medium supplemented with 10% FCS; JMSU1 cells were cultured in RPMI Glutamax medium supplemented with 10% FCS. Normal human urothelial (NHU) cells were established as finite cell lines and cultured in complete keratinocyte serum-free medium, as described in the article of Southgate et al (Southgate et al., 1994). In these experiments, two independent NHU cell lines were used at passage 4. For analyses of the effect of trichostatin A (TSA) (Calbiochem, Fontenay-sous-Bois, France) and/or 5-aza-deoxycytidine (Calbiochem) on transcript expression, normal and tumor cells were seeded in 25 cm2 dishes at a density of 8×105 cells/dish. Cultures were treated the next day with 300 nM trichostatin A (TSA) for 16 hours, 5 μM 5-aza-deoxycytidine for 72 hours, or 5 μM 5-aza-deoxycytidine for 48 hours followed by 300 nM TSA for 10 hours. These experiments were repeated twice and each time, each condition was tested in duplicate.
All bladder cancer and NHU cell lines were seeded in 12-well plates at a density of 5×104 cells/well. Cultures were treated the next day, in duplicate, with various doses of trichostatin A, from 100 nM to 500 nM, with two wells left untreated. After 72 hours, the living cells in each treated well were harvested and counted and compared to the numbers of cells in the non-treated wells. The resulting ratio was used to assess sensitivity to trichostatin A.
FGFR3 mutations were studied using the SNaPshot technique as described in Van Oers et al. (Van Oers et al., 2005).
1 μg of total RNA was used for reverse transcription, with random hexamers (20 pmol) and 200 U MMLV reverse transcriptase. To assess mRNA levels by real-time quantitative PCR (RT-qPCR), we used either individual assays or the TaqMan Low Density Array (TLDA) on an ABI PRISM 7900 real-time thermal cycler (Applied Biosystems, Foster City). With both methods, all samples were run in duplicate. For all experiments involving T1207 and CL1207 both methods were used. For individual assays, the SYBR Green kit was used to measure the expression of the RNAs of interest and the Taqman kit (Applied Biosystems) for the reference RNAs (18S rRNA). For TLDA, the same reference 18S was used; predesigned TaqMan probe and primer sets for the different genes were chosen from the Applied Biosystems catalogue. Amounts of mRNAs of the genes of interest were normalized to that of the reference gene according to the 2−ΔCt method.
The methylation status of the promoters was assessed by bisulfite sequencing and COBRA (Xiong et al., 1997). Briefly, 2 μg of genomic DNA was treated with sodium bisulfite, purified using the Epitect kit (Qiagen) and amplified as follows: initial incubation at 94° C. for 4 minutes, followed by 35 cycles of denaturation at 94° C. for 30 seconds, annealing at Tm for 30 seconds and extension at 72° C. for 30 seconds, using Biolabs Taq Polymerase (Ozyme, Saint-Quentin-en-Yvelines, France). For bisulfite sequencing, the purified PCR product was cloned using TA cloning kit (Invitrogen, Cergy Pontoise, France) and ten clones for each sample and gene were sequenced. For COBRA, the PCR products were digested for 16 hours with a restriction enzyme recognizing a restriction site containing a CpG dinucleotide. The corresponding CpG site is inferred as methylated when the PCR product is digested.
Chromatin immunoprecipitation (ChIP) assays were carried out in duplicate in three 150 cm2 dishes for untreated CL1207, CL1207 treated with 300 nM TSA for 16 h, TCCSUP, RT112 and NHU cells. Chromatin was prepared with an enzymatic kit (Active Motif, Rixensart, Belgium). An extract of the original chromatin was kept as an internal standard (Input DNA). The complexes were immunoprecipitated with 4 μg of antibodies against trimethyl histone H3 (Lys27) (Upstate Biotechnology, Santa Cruz), trimethyl histone H3 (Lys9) (Abcam, Cambridge, UK) or acetyl histone H3 (Lys9) (Abcam). The amount of immunoprecipitated target was determined by real-time PCR, in duplicate, using the ABI PRISM 7900HT Sequence Detection System. For each sample and each promoter, an average CT value was obtained for immunoprecipitated material and for the input chromatin. The amount of immunoprecipitated material was defined as 2̂(CT(Input DNA)−CT(Immunoprecipitated DNA)).
For all Affymetrix array expression analyses, Affymetrix MASS signal values were Log 2-transformed and normalized by removing chip-specific and probe set-specific effects (the mean signal for all probe sets across one chip and the mean signal for one probe set across all chips, respectively). Statistical analysis and numerical calculations were carried out with R 2.6 (R Foundation for Statistical Computing) and Amadea® (Isoft, Gif-sur-Yvette, France).
Cluster analyses were used (i) to identify, from Affymetrix expression data, regions of correlated expression, independent of copy number changes, which presented an up or downregulation in subsets of tumor samples, (ii) to identify tumors with the RES phenotype using Affymetrix (
Two different methods were used to define the RES phenotype.
(1) Using individual clustering (
(2) Using the region expression score (
By combining transcriptome and CGH array data for a set of 57 bladder carcinomas of varying grade and stage, 28 copy number-independent regions of correlated expression have been previously identified (Stransky et al., 2006). The strategy used is summarized by the example in
The inventors next investigated whether within each of the 28 regions, the correlated expression of genes was due to down and/or upregulation, and whether for each region, the deregulation was represented by all or a subset of tumors. Thus, for each region a clustering analysis of tumor and normal samples was performed according to the expression of the correlated genes, as determined by Affymetrix arrays. For each correlated gene, the ratio between its expression value in each sample and its mean expression in normal urothelium was calculated (n=5). These expression ratios were then used to cluster, for each region, all normal and tumor samples. To look for regions of downregulation (or upregulation), tumor samples with genetic losses (or gains) in these regions were excluded from the clustering analysis. This analysis identified several categories of region. For some regions, the correlated expression of genes was due to a downregulation, with this downregulation affecting only a subset of tumors. Other regions were upregulated in a subset of tumors. A third group of regions was downregulated in some tumors and upregulated in others. The remaining regions displayed no clear expression pattern. Of the 28 copy number-independent regions of correlation, seven displayed only downregulation (regions 1-1, 3-2, 3-5, 6-7, 7-2, 14-1 and 19-3). Region 19-3 could be sub-divided into two sub-regions of downregulation (19-3A and 19-3B), as cluster analysis showed that these two sub-regions were separated by 1.3 Mb which contained several genes that displayed normal expression values. Two regions (regions 2-7 and 17-7) were subjected to both down- and upregulation and six were subjected only to upregulation (1-6, 2-3, 4-2, 5-3, 6-3 and 12-4). As the inventors were interested in regions that were possibly subject to epigenetic silencing, they focused subsequent analysis on the 10 regions which presented downregulation (regions 1-1, 2-7, 3-2, 3-5, 6-7, 7-2, 14-1, 17-7, 19-3A and 19-3B).
To determine if the downregulation in these 10 regions affected stretches of contiguous genes, an extensive study of the expression of all genes within these regions was performed by RT-qPCR, analyzing both the genes present and not present on Affymetrix U95A arrays. This analysis was carried out on tumor T1207 and a cell line derived from this tumor, CL120716. Tumor T1207 was chosen because it showed downregulation in all 10 regions as shown by Affymetrix data (data not shown), and it did not present any genetic loss in these regions, as shown by CGH array (data not shown). Also, the availability of a cell line from this tumor allowed subsequent functional analyses.
Re-Expression of the Downregulated Regions Following Treatment with 5-Azadeoxycytidine and/or TSA
Tumor T1207 and its derived cell line CL1207 presented identical downregulation profiles. CL1207 was therefore used to investigate whether all genes within the nine silenced stretches were coordinately affected by an epigenetic mechanism. In particular, it was tested whether DNA methylation and/or histone acetylation/methylation might be involved. Firstly, CL1207 cells were treated with the DNA demethylating agent, 5-aza-deoxycytidine, and/or with the histone deacetylase inhibitor, trichostatin A (TSA). These different treatments led to reexpression of most of the genes in seven regions (2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B) (
In two regions (regions 6-7 and 17-7), treatment of CL1207 cells with 5-azadeoxycytidine and/or TSA led either to no re-expression or re-expression of only one isolated gene (
The Silencing of Entire Chromosomal Regions is Associated with Abnormal Histone Modification Patterns
The possible involvement of DNA methylation and/or histone hypoacetylation/methylation in the silencing of the seven regions re-expressed after treatment with 5-aza-deoxycytidine and/or TSA was investigated.
DNA methylation and histone modifications (H3K9me3, H3K27me3 and H3K9ac) were analyzed in detail for three of these regions (regions 2-7, 3-2 and 19-3A) (
The DNA methylation status of CpG islands associated with promoters was examined in tumor T1207 and its derived cell line CL1207 by bisulfite sequencing. DNA from NHU cells and fully-methylated DNA were used for comparison. The results are shown for region 2-7 (
Histone modifications in the cell line CL1207 in the promoter regions of the genes located in these three regions (regions 2-7, 19-3A, 3-2) were then investigated, using chromatin immunoprecipitation (ChIP) followed by qPCR. Antibodies specific for two inactive marks (trimethylation of Lys9 of histone H3 (H3K9me3) and trimethylation of Lys27 of histone H3 (H3K27me3)) and for one active mark (acetylation of Lys9 of histone H3, H3K9ac) were used (
DNA methylation and the same histone modifications (H3K9me3, H3K27me3 and H3K9ac) were also analyzed for the four other silenced regions (3-5, 7-2, 14-1 and 19-3B). In this case, the COBRA method (Xiong et al., 1997) was used and the DNA methylation studies were restricted to the CpG islands around promoters of the genes re-expressed after 5-aza-deoxycytidine treatment alone (
These results showed that the seven identified regions of downregulation were silenced by an epigenetic mechanism involving histone modifications. Promoter DNA methylation was very rare and when present was not significant enough to explain the silencing of these regions.
Identification of a Regional Epigenetic Silencing Phenotype Associated with Muscle-Invasive Bladder Carcinomas
The inventors have shown that the same tumor T1207 showed simultaneous epigenetic downregulation of all seven regions (2-7, 3-2, 3-5, 7-2, 14-1, 19-3A and 19-3B). In addition, cluster analysis had indicated that for each of the seven regions, downregulation was restricted to specific subsets of tumors. To determine if common silencing of the different regions occurred in the same group of bladder tumors, it was first tested whether these subsets of tumors overlapped. In
Most muscle-invasive tumors (T2-4) develop from carcinoma in situ (CIS) (Wu et al. 2005) as illustrated in
Six of the seven regions defining the RES phenotype presented H3K27 trimethylation, the footprint of the EZH2 methyl-transferase. EZH2 mRNA levels in the 57 tumors (as determined by RT-qPCR analyses) were then compared with those in normal urothelia. Nineteen of the 26 tumors with RES phenotype, but only five tumors without the RES phenotype presented a significant over-expression of EZH2 (
The existence of the RES phenotype and its association with aggressive bladder tumors of the CIS pathway was validated in an independent set of 40 bladder tumors of various stages and grades. The expression of all genes within the seven identified regions along with the genes that define the CIS signature (Dyrsjkot et al., 2004) were studied by RT-qPCR using TaqMan Low Density Array (TLDA). Twenty of the 40 tumors presented the RES phenotype (
Trichostatin a Strongly Inhibits the Growth of Bladder Cancer Cell Lines with the RES Phenotype
The findings described above have shown that the RES phenotype is associated with a subgroup of invasive tumors, and that the phenotype corresponds to the silencing of regions by H3K9 and K27 methylation and histone H3K9 hypoacetylation, but not DNA promoter methylation. TSA was used to treat a panel of bladder cancer-derived cell lines representative of the diversity of bladder tumors to determine whether the regional epigenetic silencing was restricted to a subset of bladder cancer cell lines (just as it was restricted to a subset of tumor samples). Two cell lines derived from well-differentiated tumors (MGHU3, which is mutated for FGFR3, and RT112) and four cell lines derived, like CL1207, from high-grade tumors (T24, TCCSUP, HT1376 and JMSU1, none mutated for FGFR3, and only T24 mutated for HRAS (Saison-Behmoaras et al., 1991)) were used. HRAS mutations, like FGFR3 mutations, are thought to be associated with the Ta progression pathway (
The effect of TSA was first investigated on re-expression of the genes within the seven epigenetic regions defining the RES phenotype. Re-expression results for three regions (2-7, 3-2 and 19-3A) are shown in
ChIP experiments were also carried out on three regions in detail (2-7, 3-2 and 19-3A) and for one gene in each of the other regions (3-5, 7-2, 14-1 and 19-3B) in the TCCSUP cell line, where all regions were re-expressed after TSA treatment and in RT112 cells, where no region was re-expressed, except two genes in region 7-2. For all seven regions, high levels of trimethylation of lysines 9 and 27 were observed in TCCSUP, but no significant trimethylation of either lysine 9 or 27 in RT112 (
Thus, the bladder tumor cell lines, like tumor samples (
The RES phenotype was characterized by strong histone K9 and K27 methylation and K9 hypoacetylation, but extremely rare DNA methylation. Therefore, the growth inhibiting effects of TSA—a histone deacetylase inhibitor, which indirectly inhibits histone methylation—were compared on various cell lines with and without the RES phenotype (
Using a combination of bioinformatics and experimental approaches, the inventors have defined seven chromosomal regions that can be simultaneously silenced in cancer. The silencing occurred in association with histone H3K9 hypoacetylation and H3K9 and K27 hypermethylation of promoter regions, mimicking the formation of facultative heterochromatin domains. Trichostatin A enabled gene re-expression and reversal of histone marks, clearly implicating the histone modifications in the silencing process. The demonstration that these regions were silenced simultaneously in the same set of tumors reveals, for the first time, the existence of a regional epigenetic silencing (RES) phenotype in cancer. The tumors with the RES phenotype are those tumors belonging to one of the two pathways of bladder tumor progression, the CIS pathway, which is responsible for the majority of invasive bladder tumors.
Affymetrix array expression was used to find markers for the RES phenotype. For all analyses, Affymetrix MASS signal values were Log 2-transformed and normalized by removing chip-specific and probe set-specific effects (the mean signal for all probe sets across one chip and the mean signal for one probe set across all chips, respectively). Statistical analysis and numerical calculations were carried out with Amadea® (Isoft, Gif-sur-Yvette, France). A SAM analysis (Tusher et al., PNAS 2001) was first performed between tumors with RES phenotype and invasive tumors without the RES phenotype. This analysis was restricted to the genes upregulated in the samples with RES phenotype with q-value<0.05. Genes with a fold-change above 2 was first selected. Then, the expression in the tumors with RES was compared with the normal urothelium samples and the muscle samples. Genes for which: 1) the signal was in average two times higher in the RES tumors compared to the normal samples, and 2) the signal was higher in the tumors with RES phenotype than in the muscle, were selected. 11 markers were obtained. EZH2 which was studied with RT-qPCR and found to be significantly more highly expressed in the tumors with RES phenotype was added. All markers and the expression of these markers in tumor samples compared to normal and muscle samples are presented in
150 tumors were used to study gene expression. These carcinomas were obtained from patients included between 1988 and 2001 in the prospective database established in 1988 at the Department of Urology of Henri Mondor Hospital. Four normal urothelial samples, obtained as previously described were also used for transcriptome analysis. 40 of the 150 tumor samples and three normal samples were analyzed by RT-qPCR with TLDA format (Applied Biosystems, Courtaboeuf, France). All patients provided informed consent and the study was approved by the ethics committees of the different hospitals.
RNA and DNA were extracted from the samples by cesium chloride density centrifugation. RNA and DNA were extracted from cell lines with Qiagen extraction kits (Qiagen, Courtaboeuf, France).
1 μg of total RNA was used for reverse transcription, with random hexamers (20 pmol) and 200 U MMLV reverse transcriptase. To assess mRNA levels by real-time quantitative PCR (RT-qPCR), TaqMan Low Density Array (TLDA) was used on an ABI PRISM 7900 real-time thermal cycler (Applied Biosystems). All samples were run in duplicate and the reference 18S was used. Amounts of mRNAs of the genes of interest were normalized to that of the reference gene according to the 2−ΔCt method.
Sorting Tumors with/without Regional Epigenetic Silencing (RES) Phenotype
To analyze which samples displayed the RES phenotype, the method described in example 1 was used.
mRNA levels of histone deacetylases HDAC1, 2, 3, 4, 5, 6, 7, 8 and 9 were compared between invasive tumors with and without RES phenotype. The inventors found that HDAC9 was significantly (p<0.05) over-expressed in invasive tumors with RES phenotype compared to normal samples and to invasive tumors without RES phenotype (
Patients and tissue samples were provided as described in example 3.
RNA and DNA extraction were performed as described in example 3.
Cell Culture and siRNA Transfection
The bladder cancer cell line CL1207 was cultured in DMEM F-12 Glutamax medium supplemented with 10% FCS. Cells were transfected using Lipofectamine RNAiMAX (Invitrogen) with siRNA targeted against EZH2, and a scrumble siRNA as a negative control. Gene expression analyses and ChiP experiments were carried out 80 hours after transfection. Normal human urothelial (NHU) cells were established as finite cell lines and cultured in complete keratinocyte serum-free medium, as described (De Boer et al., 1997).
1 μg of total RNA was used for reverse transcription, with random hexamers (20 pmol) and 200 U MMLV reverse transcriptase. To assess mRNA levels by real-time quantitative PCR (RT-qPCR), individual assays were used for the cell line experiments and the TaqMan Low Density Array (TLDA) was used for tumor samples, both on an ABI PRISM 7900 real-time thermal cycler (Applied Biosystems). With both methods, all samples were run in duplicate and the same reference 18S was used. Amounts of mRNAs of the genes of interest were normalized to that of the reference gene according to the 2−ΔCt method.
Chromatin immunoprecipitation (ChIP) assays were carried out as previously reported (Stransky et al., 2006) in duplicate for CL1207 cells with or without siRNA transfection. Chromatin was prepared with an enzymatic kit (Active Motif, Rixensart, Belgium). An extract of the original chromatin was kept as an internal standard (Input DNA). The complexes were immunoprecipitated with 4 μg of antibodies against trimethyl histone H3 (Lys27) (Upstate Biotechnology, Santa Cruz, USA). The amount of immunoprecipitated target was determined by real-time PCR, in duplicate.
For Affymetrix array expression analyses, Affymetrix MASS signal values were Log 2-transformed and normalized by removing chip-specific and probe set-specific effects (the mean signal for all probe sets across one chip and the mean signal for one probe set across all chips, respectively). TLDA arrays were normalized using the 18S signal and by removing the mean signal for one taqman probe across all samples and Log 2-transformed. Statistical analysis and numerical calculations were carried out with R 2.6 (R Foundation for Statistical Computing) and Amadea® (Isoft, Gif-sur-Yvette, France).
Sorting Tumors with/without Regional Epigenetic Silencing (RES) Phenotype
The sorting of tumors with and without RES phenotype was performed as described in example 1.
EZH2 mRNA expression levels was compared in a wide tumor set (n=150) between invasive tumors with (n=74) and without (n=29) RES phenotype and normal urothelium samples (n=4). Tumors with RES phenotype were identified as described above. This analysis was limited to invasive tumors in order that differences in expression levels between RES positive and negative tumors would be attributable to the phenotype itself and not the heterogeneity of tumor stages between each group.
As shown in
The role of EZH2 overexpression was studied in vitro in a cell line with RES phenotype, CL1207. CL1207 is a bladder cancer cell line derived with few passages from an invasive bladder tumor (De Boer et al., 1997). A knockdown of EZH2 was performed using siRNA. The effects of the siRNA transfection were analyzed on two chromosomal regions involved in the RES phenotype, regions 2-7 (comprising HOXD4, HOXD3 and HOXD1 genes) and 3-2 (comprising VILL, PLCD1, DLEC1 and ACAA1 genes).
EZH2 is known to catalyze the addition of a trimethyl group on H3K27. Accordingly, the level of trimethylation on H3K27 was studied by ChIP assay. Moreover, EZH2 gene expression was monitored by RT-qPCR.
Initially, when genes of regions 2-7 and 3-2 were silenced, H3K27 was highly trimethylated along these regions in comparison to the promoter of a ubiquitously expressed gene GAPDH (
These results demonstrate that the inhibition of the histone methyltransferase EZH2 induces the re-expression of genes in silenced regions involved in the RES phenotype.
Trichostatin A targets all HDACs. To narrow down the list of HDACs potentially involved in the regulation of the repressed regions, the inventors used other inhibitors specific of one or several HDACs. They found that MS275, known for its inhibition of HDAC1, 2 and 3, enabled gene re-expression in the studied regions as well as did TSA (See
To improve the list of markers allowing the discrimination of the RES phenotype of a tumor, the inventors used a larger tumor set with better-quality chips. 157 bladder tumors were studied by Affymetrix Exon arrays. First, the inventors used a clustering approach to characterize the RES status of all tumors. They clustered tumors according to the expression level they displayed in all the regions characterizing the RES phenotype. Tumors were classified in two groups, RES+ (i.e., having the RES phenotype) or RES− (i.e., not having the RES phenotype). For further analyses, the inventors only kept the invasive tumors as most RES+ tumors are already invasive. The inventors wanted to identify positive markers of RES+ and RES− tumors, i.e. markers that are over-expressed in either group compared to the other group and to normal samples. To do so, they first selected all the genes of the array that answered these criteria (over-expressed by two-fold in one of the groups compared to the other group and to normal bladder samples). Then, the inventors performed a PAM analysis to study which set of these pre-selected genes could best classify the invasive tumors according to their RES status. A set of 50 markers (see below) enabled the classification of RES+/− tumors with a minimum error rate: when studying to the entire tumor set, the error rate was 2.5% (4 errors of classification for 157 tumors). This list can be limited to the first 27 markers (see
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/61566 | 8/9/2010 | WO | 00 | 7/31/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61232496 | Aug 2009 | US |
