WO2008125883A1 - Marqueurs du cancer pour le pronostic et le criblage d'agents anticancéreux - Google Patents

Marqueurs du cancer pour le pronostic et le criblage d'agents anticancéreux Download PDF

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WO2008125883A1
WO2008125883A1 PCT/GB2008/050261 GB2008050261W WO2008125883A1 WO 2008125883 A1 WO2008125883 A1 WO 2008125883A1 GB 2008050261 W GB2008050261 W GB 2008050261W WO 2008125883 A1 WO2008125883 A1 WO 2008125883A1
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cells
cancer
drosha
mirna
mir
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PCT/GB2008/050261
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Nicholas Coleman
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Cancer Research Technology Limited
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Priority claimed from GB0707306A external-priority patent/GB0707306D0/en
Priority claimed from GB0722774A external-priority patent/GB0722774D0/en
Application filed by Cancer Research Technology Limited filed Critical Cancer Research Technology Limited
Publication of WO2008125883A1 publication Critical patent/WO2008125883A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the field of cancer diagnosis, including methods of staging and prognosis. More particularly, the invention relates to cancer markers of relevance in determining the likely outcome for a patient suffering from cancer. The markers are of prognostic value and can readily be determined from cancer cells taken, for example, from a biopsy sample. The invention also relates to the use of cancer markers in screening assays for novel anti-cancer agents.
  • MicroRNAs are a family of short, non-protein coding, RNAs that negatively regulate gene expression. The precise number is unknown though is estimated at 1000 (Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E. Cell 2005; 120(l):21-24). Forty of the 986 miRNAs in the miRNAMap database (National Chiao Tung University, Taiwan) are on chromosome 5p. The primary transcripts (pri-miRNA) are generated by polymerase II, are polyadenylated, have a 5' 7-methyl guanosine cap and a hairpin structure (Lee Y, Kim M, Han J, et al.
  • the RNase III endonuclease Drosha and its partner DGCR8 form a microprocessor that is essential for the initial stages of miRNA biogenesis (20).
  • This complex cleaves pri- miRNAs, consisting of a terminal loop, a hairpin stem and 5' and 3' single stranded RNA extensions, to pre-miRNAs (60-70-nucleotide stem loop structures) in the nucleus.
  • Pre- miRNAs are then transferred by Exportin-5 (encoded by XPO5) into the cytoplasm (21) where they are further cleaved by another RNase III endonuclease, Dicer, to generate mature ⁇ 21 -nucleotide miRNA duplexes (22).
  • RISC RNA-induced silencing complex
  • miRNA negatively regulate their mRNA targets. They may bind with complete complemetarity to their target which results in RNA cleavage by the multi-protein RNA- induced silencing complex (miRISC). This mechanism has been shown to occur in mammals (Yekta S, Shih IH, Bartel DP Science 2004; 304(5670):594-596), though it is thought to be more relevant in plants.
  • miRNAs may bind with imperfect complementarity to 3 ' untranslated regions (UTRs) of their mRNA targets. This mechanism represses target genes post-transcriptionally through a RISC complex similar to the one described above. Consistent with this notion of post- transcriptional modification, target genes regulated by this latter mechanism display decreased protein levels but not mRNA levels.
  • miRNA expression array profiles have also been shown to more accurately identify the tissue of origin of poorly differentiated tumours that their mRNA expression array counterparts (Lu J, Getz G, Miska EA, et al. Nature 2005; 435(7043):834-838).
  • miRNAs have been shown to be involved in cell growth and tissue differentiation and hence may be considered oncogenes (or tumour suppressor genes) in their own right.
  • An example is miR-21 which is expressed at a 5-100 fold higher level in glioblastoma cell lines than normal tissue (Ciafre SA, Galardi S, Mangiola A, et al. Biochem Biophys Res Commun 2005; 334(4): 1351-1358).
  • This miRNA has been shown to be an antiapoptotic factor in the same cells (Chan JA, Krichevsky AM, Kosik KS Cancer Res 2005; 65(14):6029-33.
  • Cervical carcinoma remains the second-most common cause of cancer related deaths in women worldwide, with approximately 450,000 new cases each year. The majority are squamous cell carcinomas (SCCs), which arise through precursor squamous intraepithelial lesions (SILs). Infection by high-risk human papillomaviruses (HR-HPVs) is a necessary but insufficient step for the development of cervical SCC(I), with acquisition of host genomic abnormalities also required for malignant progression. Copy -number gain and amplification of chromosome 5p occurs in 51% (mean value; range: 30%-77%) of advanced stage cervical SCCs but not in pre-malignancy(2-5), strongly suggesting an important role in progression.
  • SCCs squamous cell carcinomas
  • HR-HPVs high-risk human papillomaviruses
  • Copy -number gain and amplification of chromosome 5p occurs in 51% (mean value; range: 30%-77%) of advanced stage cervical SCCs but not
  • miRNA levels do not appear to change in only one direction during tumourigenesis at various anatomical sites (25).
  • the inventors have discovered that certain genes on chromosome 5p contribute to carcinogenesis, particularly cervical carcinogenesis.
  • the inventors used the Wl 2 model, being a cervical keratinocyte cell line infected with HPV 16, the HPV type most commonly found in cervical SCCs (1).
  • W12 recapitulates cervical neoplastic progression, both histologically and cytogenetically (9).
  • CGH comparative genomic hybridization
  • Expression microarray analysis revealed to the inventors that the most significantly upregulated transcript on 5p in W12ser-lp22 vs. W12ser-lpl9 was the RNase III enzyme Drosha, a major micro-RNA (miRNA) processing gene.
  • Drosha a major micro-RNA (miRNA) processing gene.
  • the inventors used a combination of genomic technologies to show that Drosha is frequently gained and over-expressed in carcinomas, particularly cervical SCC, where it modifies miRNA expression profiles.
  • the present invention provides a method of identifying or staging cancer in an individual comprising determining the level of RN3 RNaseIII endonuclease (Drosha) in a sample of cancer cells obtained from the individual.
  • the methods of the invention permit selection of the best available treatment regimen for an individual patient on the basis of knowing whether or not the tumour cells the patient has is going to develop into malignant and/or late stage disease.
  • the methods of the invention permit the earliest possible prognosis thereby permitting clinicians to decide on the optimum strategies for treating the cancer.
  • the prognostic methods of the invention are independent of the stage to which the cancer has developed in a patient.
  • the methods of the invention provide indications of the likelihood (or otherwise) that patient cancer has (or will have) a malignant phenotype.
  • the determination of the level of Drosha is preferably undertaken in a sample of cancerous cells taken from an individual, usually from a biopsy sample although surgically removed cancerous tissues can provide the sample materials when required.
  • the cells in which the Drosha levels are determined are preferably tumourigenic but without invasive or malignant character.
  • the Drosha determination may be undertaken on any cancer cell of whatever stage.
  • An increased level of Drosha protein or activity in the cancer cells compared to normal cells or pre-malignant cancer cells correlates with an adverse clinical outcome for the individual.
  • Such an adverse clinical outcome may be a low probability of survival in late stage disease, e.g. less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1 % chance of survival.
  • Another adverse clinical outcome for an individual may be the chances of developing late stage disease or malignancy. Such chances may be expressed as a percentage, e.g. more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99%.
  • the cancer in question is preferably one characterized by a gain of all or part of chromosome 5p.
  • Such cancers will be well known to a person of skill in the art.
  • the cancer may be one selected from cancers of the cervix, vagina, vulva, anus, lung, oesophagus, skin, head (e.g. oral cavity, larynx, tonsil, nasopharynx), neck, thymus and bladder.
  • the cancer is a squamous cell carcinoma (SCC) it is preferably an anogenital cancer selected from cervix, vagina, vulva and anus, or a cancer selected from lung, oesophagus, skin, head (e.g. oral cavity, larynx, tonsil, nasopharynx), neck, thymus and bladder.
  • SCC squamous cell carcinoma
  • the cancer is a carcinoma, preferably selected from cancer of the lung, breast, bowel, ovary, bladder, prostate, kidney, pancreas, thyroid, liver and stomach.
  • the cancer may be one selected from leukaemias, lymphomas, mesotheliomas, sarcomas, brain gliomas, childhood tumours (eg Wilms tumour, neuroblastoma).
  • the cancer is a cervical squamous cell carcinoma (SCC).
  • SCC cervical squamous cell carcinoma
  • the cancer cells may be malignant when the method of the invention is to be used for detecting malignancy.
  • an increased level of Drosha protein or activity is determined by measuring the copy number of the Drosha gene in the cells. Such methods of measurement are well known to the average skilled reader in the field.
  • the increased level of Drosha protein or activity is determined by measuring an increased level of Drosha gene transcripts in the cells compared to normal cells, pre -malignant cancer cells or cancer cells not over-expressing Drosha. This does not exclude the possibility of measuring both copy number of the Drosha gene as well as the level of Drosha gene transcripts. Such methods of measurement are well known to an average skilled reader in the field.
  • the level of Drosha protein or activity itself can be measured and quantitated to provide the necessary measurement of a relative increase in Drosha in the sample cancer cells.
  • miRNAs can be measured, but the aforementioned list represents the preferred selection of miRNA species.
  • an increased level of miRNA-31 in cancer cells compared to normal cells, pre -malignant cancer cells, or cancer cells not over-expressing Drosha correlates with (a) neoplastic progression, or (b) late stage disease, (c) malignancy and/or (d) an adverse clinical outcome for the individual.
  • a decreased level of miRNA-193b in cancer cells compared to normal cells, pre -malignant cancer cells, or cancer cells not over-expressing Drosha may correlate with (a) neoplastic progression, (b) late stage disease, (c) malignancy and/or (d) an adverse clinical outcome for the individual.
  • an increased level of miRNA-203 and/or miRNA-205 in cancer cells compared to normal cells, pre -malignant cancer cells, or cancer cells not over- expressing Drosha may correlate with (a) neoplastic progression, (b) late stage disease, (c) malignant change from normal tissue, and/or (d) an adverse clinical outcome for the individual.
  • Any of the methods of the invention described above may further comprise measuring the level of protein or activity of one or more of DGCR8, XPO5 and Dicer, and/or the level of expression or copy number gain of the genes for DGCR8, XPO5 and/or Dicer.
  • No increase in level of protein or activity, expression or copy number gain for one or more of DGCR8, XPO5 and Dicer compared to normal cells or pre -malignant cancer cells preferably correlates with (a) neoplastic progression, (b) late stage disease, (c) malignancy, and/or (d) an adverse clinical outcome for the individual.
  • the invention provides the use of RN3 RNaseIII endonuc lease (Drosha) as a marker for (a) neoplastic progression of a cancer, or (b) late stage disease of a cancer, (c) malignancy and/or (d) adverse clinical outcome for an individual suffering from a cancer.
  • Drosha RN3 RNaseIII endonuc lease
  • An increased level of Drosha in a sample of cancer cells obtained from an individual when compared to normal cells or pre -malignant cancer cells is indicative or diagnositic of (a), (b), (c) or (d) above.
  • the invention also includes the use of an miRNA transcript selected from any one or more of miR-29b, miR-492, miR-7, miR-182, miR-130a, miR-21, miR-193a, miR-365, miR-193b, miR-203, miR-34c, miR-205, miR-31 and miR-141 as a marker for (a) neoplastic progression of a cancer, or (b) late stage disease of a cancer, (c) development of malignancy, and/or (d) adverse clinical outcome for an individual suffering from a cancer.
  • an miRNA transcript selected from any one or more of miR-29b, miR-492, miR-7, miR-182, miR-130a, miR-21, miR-193a, miR-365, miR-193b, miR-203, miR-34c, miR-205, miR-31 and miR-141 as a marker for (a) neoplastic progression of a cancer, or (b)
  • An increased level of miRNA-31 in cancer cells obtained from the individual compared to normal cells, pre -malignant cancer cells, or cancer cells not over-expressing Drosha is preferably diagnostic of (a), (b), (c) or (d) above.
  • a decreased level of miRNA- 193b in cancer cells obtained from the individual compared to normal cells, pre-malignant cancer cells, or cancer cells not over-expressing Drosha may be diagnostic of (a), (b), (c) or (d) above.
  • an increased level of miRNA-203 and/or miRNA-205 in cancer cells obtained from the individual compared to normal cells, pre-malignant cancer cells, or cancer cells not over-expressing Drosha may be diagnostic of (a), (b), (c) or (d) above.
  • the invention also provides a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to test cells, the test cells having gained or gaining in culture a known and increased level of Drosha protein or activity compared to normal cells, (ii) culturing the exposed test cells for a period and then measuring the level of Drosha protein or activity in the test cells, whereby no or no significant increase in level of Drosha protein or activity in the test cells identifies an anti-cancer agent and/or an increase in level of Drosha protein or activity in the test cells identifies a poor or inactive anti-cancer agent.
  • test cells having a known and increased level of Drosha protein or activity
  • employment of such cells may permit straightforward measurement of levels of Drosha following exposure to test agent without the need for control cells.
  • Standardisation of Drosha protein or activity per cell may allow direct comparison of results before and after exposure to candidate agent.
  • the invention also includes a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to cells having gained or gaining in culture an increased level of Drosha protein or activity compared to normal cells, (ii) not exposing the candidate agent to further of said cells, (iii) culturing the exposed and the non- exposed cells for a period, (iv) measuring the levels of Drosha protein or activity in the exposed and non-exposed cells, whereby no or no significant increase in level of Drosha protein or activity in exposed cells compared to the non-exposed cells identifies an anticancer agent and/or an increase in level of Drosha protein or activity in the exposed cells compared to the non-exposed cells identifies a poor or inactive anti-cancer agent.
  • the invention provides a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to test cells, the test cells having gained or gaining in culture a known and increased level of Drosha gene transcript, protein and/or Drosha gene copy number compared to normal cells, (ii) culturing the exposed test cells for a period and then measuring the level of Drosha gene transcripts and/or Drosha gene copy number in the test cells, whereby (a) no or no significant increase in the level of transcripts and/or gene copy number identifies an anti-cancer agent and/or (b) an increase in the level of transcripts and/or gene copy number in the test cells identifies a poor or inactive anti-cancer agent.
  • a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to cells having gained or gaining in culture an increased level of Drosha gene transcript, protein and/or Drosha gene copy number compared to normal cells, (ii) not exposing the candidate agent to further of said cells, (iii) culturing the exposed and the non-exposed cells separately for a period, (iv) measuring the levels of Drosha gene transcript, protein and/or Drosha gene copy number in the exposed and non-exposed cells, whereby (a) no or no significant increase in level of gene transcripts, protein and/or gene copy number compared to the non-exposed cells identifies an anti-cancer agent and/or (b) an increase in level of transcripts, protein and/or gene copy number in the exposed cells compared to the non- exposed cells identifies a poor or inactive anti-cancer agent.
  • a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to test cells, the test cells having a known and increased level of Drosha protein or activity compared to normal cells, (ii) culturing the exposed test cells for a period and then measuring the level of Drosha protein or activity in the test cells, whereby a decrease in level of Drosha protein or activity in the test cells identifies an anti-cancer agent and/or no or no significant decrease in level of Drosha protein or activity in the test cells identifies a poor or ineffective anti-cancer agent.
  • the invention further provides a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to test cells, the test cells having a known and increased level of Drosha gene transcript, protein and/or Drosha gene copy number compared to normal cells, (ii) culturing the exposed test cells for a period and then measuring the level of Drosha gene transcripts, protein and/or Drosha gene copy number in the test cells, whereby a decrease in level of transcripts, protein and/or gene copy number in the test cells identifies an anti-cancer agent and/or no or no significant decrease in transcript level, protein level and/or gene copy number in the test cells identifies a poor or ineffective anti-cancer agent.
  • the invention also provides a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to test cells, the test cells having a known and increased level of one or more miRNAs compared to normal cells , (ii) culturing the exposed test cells for a period and then measuring the level of said one or more miRNAs, whereby a decrease in level of any of said miRNAs in the test cells identifies an anticancer agent, and/or no or no significant decrease in level of any of said miRNAsin the test cells identifies an ineffective anti-cancer agent.
  • one or more miRNAs are measured and are selected from miRNA-31, miRNA-203 and miRNA 205.
  • the invention provides a method of screening for anti-cancer agents comprising the steps of (i) exposing a candidate agent to test cells, the test cells having a known and decreased level of one or more miRNAs compared to normal cells, (ii) culturing the exposed test cells for a period and then measuring the level of said one or more miRNAs, whereby an increase in level of any of said miRNAs in the test cells identifies an anti-cancer agent, and/or no or no significant increase in level of any of said miRNAs in the test cells identifies an ineffective anti-cancer agent.
  • the miRNA is miRNA- 193b.
  • Cells having increased level of protein or activity of Drosha, increased level of Drosha gene transcripts, protein and/or increased Drosha gene copy number compared to normal cells, or increased or decreased level of miRNA preferably also have a gain in all or part of chromosome 5p. This is another measurable parameter that can be useful in the screening methods of the invention. Prevention of all or part of 5p gain may be used as a positive indicator of anti-cancer activity in a candidate agent.
  • the invention provides a method of screening for anti-cancer agents comprising the steps of preparing a cell free reaction mixture comprising Drosha and a molecule which is (a) a ligand or (b) a substrate for Drosha, wherein the substrate yields a product when subject to Drosha protein or activity; exposing the reaction mixture to a candidate agent for a period and then (a) detecting any binding of Drosha to the ligand or (b) detecting any product in the test mixture.
  • a ligand of Drosha is labeled, e.g. with a fluorescent label. The binding of Drosha for its ligand and the impact of a candidate agent on that binding can be assessed by a straightforward binding assay which measures the bound and free ligand.
  • the substrate yields a coloured or fluorescent labeled product when subjected to Drosha activity.
  • No or a decreased level of binding of Drosha to its ligand on exposure to a candidate agent may indicate an active anti-cancer agent.
  • an increase or a decrease in product on exposure to candidate agent may indicate an active anti-cancer agent, e.g. increase or decrease in fluorescence, preferably no fluorescence.
  • the substrate is a pri-miRNA, wherein on exposure to a test agent, an absence or decrease in expected pre-miRNA in the test mixture indicates an active anti-cancer agent.
  • the substrate is a pri-miRNA and, wherein on exposure to a test agent an increase in expected pre-miRNA in the test mixture indicates an active anticancer agent.
  • the test (including any control) mixture may further comprise DGCR8.
  • a selected pri-miRNA has its corresponding miRNA upregulated in 5p gain cells and wherein an absence or no significant amount of pre-miRNA in the test reaction mixture after exposure to the candidate agent identifies an effective anti-cancer agent.
  • control reaction mixture not exposed to candidate agent for the period, whereby a lesser amount of pre-miRNA in the test mixture compared to the control identifies an effective anti-cancer agent.
  • the pri-miRNA is selected from one or more of pri-miRNA-31 , pri- miRNA-203, pri-miRNA-34c, pri-miRNA-141 and pri-miRNA 205.
  • a selected pri-miRNA may have its corresponding miRNA downregulated in 5p gain cells and wherein an amount of pre -miRNA detected in the test reaction mixture after exposure to the candidate agent identifies an effective anti-cancer agent.
  • the control reaction mixture is not exposed to candidate agent for the period, whereby a greater amount of pre -miRNA in the test mixture compared to the control identifies an effective anti-cancer agent.
  • the pri-miRNA is selected from one or more of pri-miRNA- 193b, pri-miRNA-365, pri-miRNA- 193 a, pri-miRNA-21 , pri-miRNA- 130a, pri-miRNA- 182, pri-miRNA-7, pri-miRNA-492, pri-miRNA-29b, preferably pri-miRNAl 93b.
  • the cell free assay mixture may further comprise Dicer in which case, following exposure of the mixture to a candidate agent for a period, the presence of any miRNA is then detected.
  • the miRNA to be detected is selected from any one or more of miR-29b, miR-492, miR-7, miR-182, miR-130a, miR-21, miR-193a, miR-365, miR- 193b, miR-203, miR-34c, miR-205, miR-31 and miR-141.
  • the invention also provides a method of treating or preventing cancer in an individual comprising administering an effective amount of an interfering RNA (RNAi) cognate to the gene encoding Drosha.
  • RNAi interfering RNA
  • the invention provides the use of interfering RNA (RNAi) cognate to the gene encoding Drosha for the manufacture of a medicament for the treatment or prevention of cancer.
  • RNAi interfering RNA
  • the medicament is preferably for the prevention of malignancy.
  • the cancer is cervical cancer, preferably squamous cell cervical cancer (SCC).
  • SCC squamous cell cervical cancer
  • RNAi small interfering RNA
  • siRNA molecules may be prepared by any suiable method, including chemical synthesis, in vitro transcription, using siRNA expression vectors and using PCT expression cassettes.
  • the design of siRNAs may include the steps of (a) finding 21nt sequences in the target mRNA that begin with an AA dinucleotide. Two, three or four target sequences may be chosen. Then, select an appropriate control, e.g. a negative control siRNA with the same nucleotide composition as the chosen siRNA but which lacks significant homology to the genome in question. Also, a positive control may be used which would comprise and additional siRNA sequence targeting the same mRNA.
  • an appropriate control e.g. a negative control siRNA with the same nucleotide composition as the chosen siRNA but which lacks significant homology to the genome in question.
  • a positive control may be used which would comprise and additional siRNA sequence targeting the same mRNA.
  • siRNA design tool is available online from the Whitehead Institute of Biomedical Research at MIT (http://jura.wi.mit.edu). Suitable siRNAs are commercially available from Ambion Inc. or Quiagen Inc.
  • a medicament of the invention is for the prevention of malignancy, preferably in instances where the cancer is cervical cancer, preferably squamous cell cervical cancer (SCC).
  • SCC squamous cell cervical cancer
  • Figure 1 a) Array-CGH view of chromosome 5 for (i) W12ser-lpl9 and (ii) W12ser- Ip22, showing 5p gain in the latter, b) A representative metaphase spread of W12ser- Ip22 hybridized with whole chromosome 5 paint (red), showing three copies of normal chromosome 5 (green arrowheads) and two copies of isochromosome 5p (yellow arrowheads), c) and d), Organotypic raft culture showing representative images of c) (i) and (ii) W12serl-pl9 and d) (i) and (ii) W12serl-p22. Both form dysplastic epithelia, with W12ser-lp22 reproducibly showing an ability to invade collagen (arrows).
  • Figure 2 Array-CGH and quantitative real-time PCR expression data for Drosha, DGCR8, XPO5 and Dicer, a) Genomic copy -number (as determined by array-CGH) in ten cervical SCC cell lines and 36 cervical SCC clinical samples. Thresholds were set at 1.2 for gain (solid line) and 0.8 for loss (dashed line), based on normal:normal hybridizations, b) Comparison of expression levels with gene copy-number in (from left to right on x axis): 10 cervical SCC tumour samples; 10 cervical SCC cell lines; four normal cervical keratinocyte primary cultures; and W12ser-lpl9 and W12ser-lp22. Array-CGH ratios are in blue, with threshold for copy-number gain (1.2) shown as a solid line. Expression Pfaffl ratios are in red, with threshold for over-expression (2.0) shown as a dashed line.
  • FIG. 3 miRNA profiles in cervical SCC depend on Drosha expression status. Note that the key is applicable to all three panels, a) (i) Principal components (PC) 2 and 3 for 282 miRNAs in cervical cell lines and clinical samples. (U) Dendrogram showing hierarchical clustering of cervical cell lines and clinical samples based on the profiles of the 14 selected miRNAs. In the heat map, red and blue indicate over- and under- expression compared to mean miRNA expression levels. (Ui) Generalized log-ratios (M- values) for four miRNAs found to be significantly up- or down-regulated in both SCC cell lines and SCC clinical specimens with Drosha over-expression (two-sided moderated t-test, adjusted p ⁇ 0.05). Circles represent M-values for four replicate probes per array.
  • PC Principal components
  • U Dendrogram showing hierarchical clustering of cervical cell lines and clinical samples based on the profiles of the 14 selected miRNAs. In the heat map, red and blue indicate over- and under- expression compared to mean miRNA expression
  • Figure 4 TaqMan quantitative real-time PCR analysis of selected miRNAs.
  • (i) and (U) Validation of microarray data (dashed bars) by real time PCR (solid bars) for miRNAs in: (i) SCC cell lines HT3 (no Drosha over-expression) and CaSki (Drosha over-expression) and (ii) clinical SCC samples T34 (no Drosha over-expression) and T55 (Drosha over- expression).
  • Figure 5 Array-CGH data for a) W12ser-lpl9 and b) W12ser-lp22. Both samples show chromosome 1Op loss and X chromosome gain (a positive internal control through the use of male reference DNA). The only difference between the samples is the presence of 5p gain in W12ser-lp22.
  • FIG. 6 (Q)PCR expression data for Drosha (RN3) and Dicer in a range of cancer cell lines. The expression levels are shown as a Pfaffl/aCGH ratio.
  • Figure 7 Heatmap and dendrogram of miRNA expression in cervical SCC cell lines and normal cervical cancer cell lines.
  • Figure 8 Volcano plot and Table of differentially expressed miRNAs between SCC + RN3 (group G) and SCC - RN3 (group C).
  • Figure 9 Volcano plot and Table of differentially expressed miRNAs between SCC - RN3 (group C) and NCx (group N).
  • Figure 10 Volcano plot and Table of differentially expressed miRNAs between SCC + RN3 (group G) and NCx (group N).
  • Figure 11 Over-expressed transcripts on 5p associated with copy-number gain.
  • Figure 12 Array CGH and qRT-PCR expression data for Drosha.
  • Figure 13 Location of siRNA recognition sites and Drosha knockdown.
  • Figure 14 Proliferation and colony forming efficiency following knockdown of Drosha.
  • Figure 15 Motility of SCC cell lines following knockdown of Drosha.
  • Figure 16 Drosha knockdown, with both pooled and individual siRNA duplexes leads to inhibition of cell invasion.
  • Figure 17 Proliferation of SCC cell lines in decreased following miR31kd but not increased following miR31 over-expression.
  • Drosha copy -number gain was seen in 21/36 clinical samples and 8/10 cell lines and there was a significant association between Drosha transcript levels and copy-number gain.
  • Drosha over-expression in cervical SCC is of functional significance.
  • Unsupervised principal component analysis of a mixed panel of cervical SCC cell lines and clinical specimens showed clear separation according to Drosha over-expression. miRNAs most significantly associated with Drosha over- expression are implicated in carcinogenesis in other tissues.
  • Drosha over-expression Many miRNAs deregulated in samples with Drosha overexpression and are associated with carcinogenesis in other tissues. Copy number driven over-expression of Drosha and subsequence changes in miRNA profiles are elements of the selective advantage provided by 5p gain in cervical neoplastic progression.
  • Tissue Samples All tissue specimens were used with Local Research Ethics Committee approval. Each sample was from a different patient. The specimens used were: (i) Cervical SCCs from the archives of the Kidwai Memorial Institute of Oncology, Bangalore, India. Tumours were staged according to the International Federation of Gynaecology and Obstetrics criteria for cervical carcinoma (http://www.figo.org/). Frozen tissue samples were from 36 pre-treatment SCCs, all of which were grade 3 and high stage (IIIA and above), (ii) Lesional epithelium microdissected from frozen sections of 25 cases of high-grade SIL and 15 cases of low-grade SIL, as previously described (11).
  • microdissected tissue was composed of abnormal epithelium, (iii) Samples of microdissected normal ectocervical epithelium, obtained from hysterectomy specimens for disease unrelated to the cervix (11). The epithelium was histologically normal and negative for HPV DNA by nested PCR and reverse line blot hybridization (11). In total, seven samples were used for array analysis.
  • the cervical keratinocyte cultures used were as follows: Four different primary cultures of normal cervical keratinocytes (NCxI, NCx2, NCx5 and NCxI l) generated from hysterectomy samples for disease unrelated to the cervix; Wl 2 series- 1 pass-19 (W12ser-lpl9) and W12 series-1 pass-22 (W12ser-lp22)(10); the cervical SCC cell lines SiHa, CaSki, C33a, C4I, C4II, SW756, MS751, HT3, DOTC2 and ME180 (all from American Type Culture Collection). Published protocols are used for cell culture, metaphase preparation, determination of colony forming efficiency and organotypic raft culture (9, 10).
  • Chromosome- Specific Paint Chromosome 5 specific paint was prepared by degenerate oligonucleotide-primed PCR from flow sorted chromosomes and labelled with digoxigenin- 11 -dUTP (Roche) (12). After overnight hybridization, bound probe was detected using anti-digoxigenin FITC-conjugated Fab fragments (Roche), counterstained with DAPI/antifade counterstain. Images were captured as described (12).
  • Array-CGH Array platforms were constructed from 4134 optimized BAC clones covering the human genome. The mean gap size was 0.92 Mb (13). Genomic DNA (gDNA) extraction, labelling and hybridization were performed as previously described (13).
  • gDNA was amplified by DOP-PCR as described (11).
  • SCC samples the gDNA was not amplified.
  • 1 ⁇ g each of test and reference gDNA (the latter from normal male peripheral blood lymphocytes) were labelled with Cy3-dCTP or Cy5-dCTP using random-priming, with dye-swapping (BioPrime Plus, Invitrogen).
  • the median of untransformed foreground pixel intensities were background corrected using the median local background.
  • the test:reference copy number ratio of corrected spot intensities were then log (base 2) transformed and a global median normalization applied. Finally, the within-array normalized log-ratios were averaged over dye-swap paired experiments.
  • Thresholds were set at 1.2 for copy -number gain and 0.8 for copy-number loss, based on three standard deviations of the mean of log-ratios in four normal:normal hybridizations (using gDNA from normal ectocervical epithelium or normal female placenta versus gDNA from normal male peripheral blood lymphocytes).
  • Quantitative Real-Time PCR Transcript levels were measured in monolayer cells and clinical samples using QuantiTect in one-step SYBR-Green RT-PCR reactions (Qiagen). Primer efficiencies were determined using 7-point serial dilutions of Universal Human Reference RNA (Stratagene). All reactions (including non-RT and no template controls) were run in triplicate using an Opticon-2 cycler (MJ Research). Fluorescence was measured at the last step of each cycle and melting curves confirming single PCR products were obtained after each run.
  • Expression ratios were calculated using the comparative Ct method described by Pfaffl (14). Values were normalized using four housekeeping genes: beta-actin, glyceraldehyde-3 -phosphate dehydrogenase, hydroxymethylbilane synthase and TATA box binding protein. Pooled RNA from four primary cultures of normal cervical keratinocytes was used as the reference sample. Based on the Pfaffl ratios observed (see Results), Drosha over-expression was defined as an expression fold-change greater than 2.0. Correlation between Drosha transcript over-expression and copy-number gain was examined using a one-sided Fisher's Test.
  • RNA-linker p-rUrUrUdA-Cy-dye (Dharmacon), labelled at the 3 '-end with Cy3 (test RNA) or Cy5 (reference RNA), using T4 RNA ligase overnight at 37°C, as described elsewhere (15). Unbound nucleotides/RNA-linkers were removed by ethanol precipitation.
  • miRNA Microarray Data Analysis were performed using the statistical programming language R and functions available from Bioconductor. Mean Cy3 and Cy5 signal intensities were read into R. Poor spots, as reported in the raw data file, and probes not annotated as miRNAs were given spot quality weight zero. The non- background corrected signal intensities were normalized using vsn (16). Further analysis was based on the generalized log (base2) ratios (M-values). Differential expression was assessed using a moderated t-statistic, taking into account spot quality weights and correlation between within-array replicate spots (17). P-values were adjusted for multiple testing using Benjamini and Hochberg's method (18).
  • PCA principal component analysis
  • hierarchical clustering For principal component analysis (PCA) and hierarchical clustering, the two data sets (cell lines and clinical samples) were pre-processed individually. Non-zero weight replicate spots were averaged and probes were mean centred. Probes with all replicate spots of zero weight for a given array were removed. No further pre-processing was undertaken prior to combining the data sets. Unsupervised PCA was performed using all 298 probes (282 miRNAs) in the combined data set. Hierarchical clustering was performed with Pearson correlation and average linkage, based on miRNAs selected for differential expression between any of the three groups of interest in cell lines (adjusted p ⁇ 0.05, moderated t-test).
  • NCxC microdissected normal ectocervical tissue sample C
  • the samples fell into two distinct groups according to Pfaffl ratios for Drosha mRNA levels, with ratios ranging from 2.86-7.67 in one group and from 0.70-1.29 in the other group.
  • a Pfaffl ratio cut-off value of 2.0 was used to define Drosha over-expressing and non over-expressing samples.
  • Over-expression of DGCR8, XPO5 and Dicer occurred infrequently and there was no correlation with copy-number (see Fig. 2b).
  • Drosha over-expression was measured in relation to miRNA expression profiles in cervical SCCs.
  • Combined miRNA expression data for ten SCC cell lines, two primary cultures of normal ectocervical keratinocytes, two passages of Wl 2 and eight clinical specimens were subjected to unsupervised PCA. Principal components 2 and 3, accounting for 24% of the variation in the combined data, showed clear separation between samples with and without Drosha over-expression [see Fig. 3a(i)].
  • Table 1 below shows miRNAs associated with Drosha over-expression in both SCC cell lines and SCC clinical samples. The estimated fold-changes are between samples over- expressing Drosha and samples not over-expressing Drosha.
  • hsa-mir-29b [MIMATOOOO 100] UAGCACCAUUUGAAAUCAGUGUU SEQ ID NO : 1 hsa-miR-492 [MIMAT0002812]
  • pre-miRNAs suitable for use as prognostic and/or diagnostic markers in accordance with the invention include:
  • DGCR8, XPO5 and Dicer did not display similar genomic gain or over-expression, suggesting that other members of the miRNA processing machinery are not responsible for the changes in miRNA profiles seen.
  • miRNAs control critical functions across various biological processes, one of which is carcinogenesis (25). miRNA fingerprinting profiles distinguish between cancers from different lineages and within a single lineage (25), although the mechanisms by which such profiles change in neoplastic tissue are poorly understood. Without wishing to be bound by any particular theory, the inventors suggest that Drosha over-expression disturbs the process of miRNA biogenesis and represents one of the mechanisms by which miRNA profiles alter during carcinogenesis. Unsupervised PCA revealed that a large amount of variation in miRNA expression profiles in cervical SCCs can be explained by Drosha over-expression. After multiple testing correction, 16 miRNAs were identified as significantly differentially expressed according to Drosha over- expression in the SCC cell lines, of which four were also significantly differentially expressed in the clinical samples.
  • miR-31 showed a mean fold-change increase in samples over-expressing Drosha (compared to cases not over-expressing Drosha) of 5.71 in clinical specimens and 8.75 in cell lines.
  • miRNAs may either increase or decrease when Drosha is over-expressed. Without wishing to be bound to any particular mechanism or theory, the inventor's suggest some reasons for this. Firstly, some pri-miRNAs may be more sensitive to changes in Drosha levels than others.
  • adenosine deaminases that act on RNAs ADARs
  • ADARs may edit pri-miRNAs in a site-specific manner (29) resulting in their degradation by Vietnamese-SN, a component of RISC (30), preventing Drosha/DGCR8 processing and mature miRNA formation.
  • ADARs convert adenosine to inosine in double stranded RNA.
  • pri-miRNAs are more susceptible to editing by ADARs than others. If so, only those miRNAs whose primary transcript is not edited would show increased levels upon Drosha over-expression. In addition, as yet undiscovered mechanisms of pri-miRNA modification may also contribute to selective Drosha/DGCR8 cleavage.
  • Drosha/DGCR8 cleavage Differences in the size of the terminal loop (31) and properties of the single-stranded RNA segments at the base of the stem of the pri-miRNA (31 , 32) have been found to alter the efficiency of Drosha/DGCR8 cleavage. Certain pri-miRNAs may have terminal loops and single-stranded stems that allow them to be cleaved more efficiently by Drosha/DGCR8. If so, a greater quantity of the respective mature miRNA products may be generated when Drosha is over-expressed.
  • miRNAs may decrease the transcription of, or degrade the mRNA of, transcription factors involved in the production of other pri-miRNAs. Since the production of different pri-miRNAs may be controlled by different transcription factors, the increase of certain mature miRNAs (by mechanisms such as those described above) may result in decreased levels of other miRNAs, by suppressing essential transcription factors required for the production of their relevant pri-miRNAs.
  • a cell free method is used for testing the activity of Drosha in producing miRNAs from selected pri-miRNAs.
  • HEK293T cells transfected with Drosha-Flag expression plasmid were harvested 2 d after transfection.
  • Total cell extract was prepared in buffer A (20 mM Tri-HCl at pH 8.0, 500 mM NaCl, 0.2 mM EDTA, 0.2 mM PMSF) by sonication followed by centrifugation.
  • the whole-cell extract was incubated with anti-Flag M2 affinity gel (Sigma) at 4°C for 2 h.
  • the Flag immunoprecipitates were drained and incubated with 1 mL of buffer B (20 mM Tris-HCl at pH 8.0, 2.5 M NaCl, 0.2 mM EDTA, 0.2 mM PMSF, 1% Triton X-100) at 4°C for 1 h in order to detach the Drosha- interacting proteins from Drosha-Flag complex.
  • the beads were washed five times with buffer B and twice with buffer A. After washing, the immunoprecipitates were drained and used for in vitro processing with 1.5 ⁇ g of recombinant proteins (GST or GST-DGCR8).
  • DGCR8 ( ⁇ N275)
  • the partial length (276-773 amino acids) of human DGCR8 cDNA was amplified from HEK293T cDNA by PCR (forward primer: 5'- GAATTCGATGGAGAGACAAGTGTGCAGC-S', reverse primer: 5'-
  • DGCR8 GCGGCCGCTCACACGTCCACGGTGCACAG-3'
  • pGEM-T easy vector Promega
  • the human DGCR8 partial-length cDNA was subcloned into pGEX-4T3 vector (Amersham), which has GST-tag at the N terminus, using EcoRI and Notl sites.
  • the expression clone of DGCR8 was transformed to E. coli BL21 -RIL strain. The expression and purification of recombinant DGCR8 were conducted according to the manufacturer's protocol.
  • RNA interference RNA interference
  • ANKH ANKH
  • BASP BASP
  • SRD5A1 SRD5A1 over-expression was investigated using RNA interference (RNAi) to transiently deplete levels in both CaSki and SW756 cells, followed by phenotypic assessment.
  • RNAi RNA interference
  • the location of individual siRNA recognition sites (#l-#4) within the Drosha open reading frame is shown in Figure 13 a.
  • Target transcripts were depleted following transient transfection of transcript specific siRNA with LipofectamineTM 2000 (Invitrogen), according to the manufacturer's instructions. The longevity of any effect was assessed using qRT-PCR at 1 day intervals. Similar levels of depletion were achieved in both CaSki and SW756 cells. Knockdown levels for Drosha are depicted in Figure 3b. Approximately 70% depletion of Drosha mRNA was achieved between 1-3 days post transfection and this time window was chosen for subsequent experiments. Individual siRNA duplexes were assessed for their ability to inhibit the levels of Drosha, in both CaSki and SW756 cells; duplexes #2 and #3 demonstrated similar efficacy to the pooled duplexes, 48 hours post transfection (Figure 13c).
  • Drosha was over-expressed in SW756 cells, using a silent mutant that was resistant to siRNA duplex #3.
  • An SW756 transcript was established that expressed this form of Drosha at levels 2.6-fold greater than wild type SW756 cells and cells transfected with the empty pcDNA3.1 vector.
  • the over-expressing population (Drosha mut3) was susceptible to duplex #2 but resistant to duplex #3.
  • Drosha mut3 cells were less invasive when treated with duplex #2 (i.e. when Drosha was depleted) but not so when treated with duplex #3 (i.e. Drosha not depleted), whereas control cells transfected with the empty vector showed a decrease in their invasive capacity on treatment with either duplex #2 or duplex #3 ( Figure 16c).
  • Advanced-stage cervical carcinomas are defined by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm 3q. Genes Chromosomes Cancer 1997;19(4):233-40.

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Abstract

L'endonucléase RN3 ARNase III (Drosha) est un composant de la biogenèse des microARN (miARN). La Drosha induit des changements dans le profil d'expression du miARN des cellules du carcinome malpighien (CM) cervical. La présente invention concerne des procédés pour identifier ou évaluer l'avancement du cancer chez un individu qui comprend la détermination du niveau de Drosha dans un échantillon de cellules cancéreuses obtenues chez l'individu. Un niveau élevé de Drosha dans l'échantillon de cellules cancéreuses indique une issue clinique négative pour l'individu. Les dosages pour le criblage d'agents anticancéreux modulent l'expression de Drosha et/ou de ces miARN dont le niveau d'expression est modulé par le niveau d'expression de Drosha.
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WO2009059026A1 (fr) * 2007-10-30 2009-05-07 Veridex, Llc Procédé de surveillance du cancer colorectal
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WO2010014975A3 (fr) * 2008-08-01 2010-05-06 Academia Sinica Utilisation de signatures en micro-arn pour évaluer des niveaux de risque de neuroblastome
WO2010083464A3 (fr) * 2009-01-16 2011-05-12 Cepheid Procédés de détection de cancers du col de l'utérus
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CN106119393B (zh) * 2016-08-25 2020-04-07 朱伟 一种与食管鳞癌辅助诊断相关的血浆miRNA标志物及其应用
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