WO2009109952A2 - Détection et traitement d'un phénotype de cancer invasif - Google Patents

Détection et traitement d'un phénotype de cancer invasif Download PDF

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WO2009109952A2
WO2009109952A2 PCT/IE2009/000005 IE2009000005W WO2009109952A2 WO 2009109952 A2 WO2009109952 A2 WO 2009109952A2 IE 2009000005 W IE2009000005 W IE 2009000005W WO 2009109952 A2 WO2009109952 A2 WO 2009109952A2
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protein
cells
aldhlal
clone
stipl
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PCT/IE2009/000005
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WO2009109952A3 (fr
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Naomi Walsh
Norma O'donovan
Paul Dowling
Martin Clynes
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Dublin City University
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01036—Retinal dehydrogenase (1.2.1.36)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the invention relates to methods of detection, inhibition, prevention, or treatment of invasive/metastatic cancer in an individual.
  • the invention relates to a methods of detection, inhibition, prevention, or treatment of invasive/metastatic cancer in an individual having pancreatic cancer.
  • Pancreatic cancer is one of the most lethal cancers and is the 8 th leading cause of cancer- related deaths in Europe (1). Pancreatic cancer is associated with poor prognosis, whereby the rate of mortality is similar to that of the rate of incidence. It is the most fatal malignancy; all-stage 5-year survival rate is less than 5% (2),(3). Conventional approaches including, surgery, radiation, chemotherapy and combination of theses therapies, has had little effect on the survival rate of patients diagnosed with pancreatic cancer. Pancreatic cancer appears to be inherently resistant to a wide variety of chemotherapeutic agents, which can differ greatly and are unrelated with respect to molecular structure and target specificity. The malignant progression of the invasiveness and metastatic potential of this cancer is complex and poorly understood.
  • pancreatic cancer In this study, we established the proteomic profile of proteins secreted into the media from pancreatic cancer cell lines with varying invasive and malignant transformation characteristics. Theoretically, proteins secreted by tumour cells are more likely to be detected easily in bodily fluids such as urine, blood serum and pancreatic ductal juice. Therefore, secreted proteins and their metabolites found in vivo could represent a panel of potential biomarkers. As pancreatic cancer invades and metastasises at an early stage without symptoms, it is vital to develop early detection systems for the diagnosis of pancreatic cancer. Studies have reported proteomic analyses of pancreatic tissue, pancreatic juice as well as blood plasma and sera (4).
  • Molecular markers and biomarkers constitute major targets for the early detection of cancer, identification of cancer risk and/or prediction of therapeutic response (5).
  • Proteomics provides an excellent means for analysis of bodily fluids for classifying proteins and identifying biomarkers for early detection of cancers.
  • the main biomarker currently available for pancreatic cancer detection, CA 19-9 has demonstrated to have sensitivity up to 90% and specificity up to 98% in the diagnosis of this malignancy (6, 7), however, this marker is not fully specific as false-positive or false- negative findings occurs in patients with other gastrointestinal malignancies and also in patients with benign disease, particularly when associated with obstructive jaundice or cirrhosis, which may contribute to late diagnosis of pancreatic cancer.
  • CA 19-9 expression will be falsely low even in the presence of advanced pancreatic cancer (9).
  • the invention is based on the finding that the expression level of certain proteins is modulated in cancers according to the invasiveness/metastatic potential of the cancer.
  • certain proteins have been found to be overexpressed in a highly invasive cancer, certain protein have been found to be underexpressed in a highly invasive cancer, and certain proteins have been found to be overexpressed in a low invasiveness cancer.
  • the expression levels of these proteins function as biomarkers of invasiveness/metastases potential, and biomarkers in the early diagnosis of cancer.
  • the invasiveness of a cancer may be attenuated by modulation of the expression of a subset of specific biomarkers, especially STIPl and ALDHlAl.
  • a method of assessing the status of a cancer in an individual comprising a step of assessing a biological sample from the individual for the expression level of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl, KRT18, GAPDH, GSN, Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6, and correlating the expression level of the protein with cancer status.
  • the GSN protein is GSN isoform b.
  • the method comprises a step of assessing a biological sample obtained from the individual for the expression level of a protein selected from the group ALDHlAl, VIM, STIPl , TPIl.
  • the method comprises a step of assessing a biological sample obtained from the individual for the expression level of a protein selected from the group KRTl 8, GAPDH, GSN.
  • the method comprises a step of assessing a biological sample obtained from the individual for the expression level of a protein selected from the group Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6.
  • cancer status should be taken to mean cancer diagnosis, especially early cancer diagnosis, invasive/metastases potential of a cancer, assessment of likely patient outcome due to the cancer, and assessment of effectiveness of a treatment for a cancer.
  • cancer should be taken to mean any cancer, including a cancer selected from the group consisting of: fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcom; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma; lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumor; leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma; bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma; chori
  • the cancer is pancreatic cancer.
  • biological sample may be any sample obtained from an individual such as, for example, blood, serum, saliva, urine, cerebrospinal fluid, tissue, cells, etc.
  • the biological sample will be serum.
  • GSN is known to be serum proteins and have been shown to be differentially expressed in the conditioning medium of cancers of differing invasiveness/metastases potential.
  • the individual will be a person suspected of having cancer, or pre-disposed to developing cancer as determined by other phenotypic, genotypic or hereditary traits.
  • the individual may be a person known to have cancer, and who is undergoing a therapeutic treatment regime, in which case the method of the invention may be employed to monitor the effectiveness of the treatment, or may be a post-operative patient being monitored for re-occurrence of the disease.
  • the method is a method of assessing the invasive/metastatic potential of a cancer, and in which overexpression of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl is associated with an invasive/metastatic potential.
  • a protein selected from the group ALDHlAl, VIM, STIPl, TPIl is associated with an invasive/metastatic potential.
  • the group of proteins comprises STIPl and ALDHlAl .
  • the method is a method for the early detection of a cancer, in which overexpression of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl is associated with early detection of the cancer.
  • the cancer is pancreatic cancer.
  • the group of proteins comprises STIPl and ALDHlAl.
  • the method is a method of monitoring the effectiveness of a treatment for a cancer, especially a treatment for reducing the invasiveness/metastates potential of a cancer, or a treatment for a metastases, in which a decrease in the expression of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl is associated with effectiveness of the treatment.
  • the group comprises STIPl and ALDHlAl .
  • the method is a method of assessing the invasive/metastatic potential of a cancer, and in which underexpression of a protein selected from the group KRT 18, GAPDH, GSN is associated with an invasive/metastatic potential.
  • the method is a method for the early detection of a cancer, in which underexpression of a protein selected from the group KRT 18, GAPDH, GSN is associated with early detection of the cancer.
  • the cancer is pancreatic cancer.
  • the method is a method of monitoring the effectiveness of a treatment for a cancer, especially a treatment for reducing the invasiveness/metastates potential of a cancer, in which an increase in the expression of a protein selected from the group KRT 18, GAPDH, GSN is associated with effectiveness of the treatment.
  • the method is a method of assessing the invasive/metastatic potential of a cancer, and in which overexpression of a protein selected from the group Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6 is associated with non- invasive/non-metastatic potential.
  • the method is a method of monitoring the effectiveness of a treatment for a cancer, especially a treatment for reducing the invasiveness/metastases potential of a cancer, in which an increase in the expression of a protein selected from the group Integrin ⁇ l, Integrin ⁇ 5, and Integrin ⁇ 6 is associated with effectiveness of the treatment.
  • the term "overexpression" of a protein should be taken to mean a level of expression of the protein which is significantly higher than the level of expression the protein in a reference non-aggressive pancreatic cancer cell.
  • the term “underexpression” of a protein should be taken to mean a level of expression of the protein which is significantly lower than the level of expression the protein in a reference non-aggressive pancreatic cancer cell.
  • the invention also relates to a method of treating a cancer in an individual comprising a step of attenuating an activity of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl in the individual.
  • the group comprises STIPl and ALDHlAl.
  • the method is a method of inhibiting, preventing or treating an invasive/metastatic cancer in an individual, typically in an individual with an established cancer.
  • the established cancer is pancreatic cancer.
  • the term "inhibiting, preventing or treating an invasive/metastatic cancer” should be understood as including one or more of decreasing the invasiveness/metastatic potential of the cancer, inhibiting or preventing invasion of the cancer cells, and preventing or treating metastases in an individual.
  • invasive/metastatic cancer should be understood as meaning invasive cancer, or metastatic cancer, or, in one embodiment, a cancer having both and invasive and metastatic phenotype.
  • the invention also relates to the use of agent capable of attenuating the activity of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl as a medicament.
  • the invention also relates to a pharmaceutical composition comprising an agent capable of attenuating the activity of a protein selected from the group ALDHlAl, VIM, STIPl, TPIl.
  • the invention also relates to a method of treating a cancer in an individual comprising a step of increasing an activity of a protein selected from the group KRT 18, GAPDH, GSN in the individual.
  • the method is a method of decreasing the invasiveness/metastatic potential of the cancer, or preventing or treating metastases in an individual.
  • the invention also relates to the use of agent capable of increasing the activity of a protein selected from the group KRTl 8, GAPDH, GSN as a medicament.
  • the invention also relates to a pharmaceutical composition comprising an agent capable of increasing the activity of a protein selected from the group KRTl 8, GAPDH, GSN.
  • the invention also relates to a method of treating a cancer in an individual comprising a step of increasing an activity of a protein selected from the group Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6 in the individual.
  • the method is a method of decreasing the invasiveness/metastatic potential of the cancer, or preventing or treating metastases in an individual.
  • the invention also relates to the use of agent capable of increasing the activity of a protein selected from the group Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6 as a medicament.
  • the invention also relates to a pharmaceutical composition comprising an agent capable of increasing the activity of a protein selected from the group Integrin B 1 , Integrin ⁇ 5, and Integrin ⁇ 6.
  • RNA interference is an evolutionally highly conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (known as siRNA molecules), when introduced into a cell, causes sequence-specific degradation of mRNA sequences.
  • RNAi machinery once it finds a double-stranded RNA molecule, cuts it up, separates the two strands, and then proceeds to destroy RNA molecules that are complementary to one of those segments, or prevent their translation into proteins.
  • suppression of a proteins expression may be achieved by treating an individual with siRNA molecules designed to target mRNA for the protein.
  • siRNA molecules designed to knockdown STIPl are provided in SEQUENCE ID NO's: 8, 9 and 10.
  • siRNA molecules designed to knockdown ALDHlAl are provided in SEQUENCE ID NO's: 11, 12 and 13.
  • Other types of gene knockdown tools will be well known to the person skilled in the filed of molecular biology.
  • miRNAs are small ( ⁇ 22nt) non-coding RNAs (ncRNAs) that regulate gene expression at the level of translation. Each miRNA apparently regulates multiple genes and hundreds of miRNA genes are predicted to be present in mammals. Recently miRNAs have been found to be critical for development, cell proliferation and cell development, apoptosis and fat metabolism, and cell differentiation.
  • small hairpin RNA (shRNA) molecules are short RNA molecules having a small hairpin loop in their tertiary structure tha may be employed to silence genes.
  • the design of miRNA or shRNA molecules capable of silencing a given protein will be apparent to those skilled in the field of miRNA or shRNA molecule design.
  • the level of protein expression can be modulated using antisense or ribozyme approaches to inhibit or prevent translation of the protein mRNA transcripts or triple helix approaches to inhibit transcription of the gene for the protein.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA for the protein. The antisense oligonucleotides will bind to the complementary mRNA transcripts and prevent translation.
  • Ribozyme molecules designed to catalytically cleave mRNA transcripts of a given protein can also be used to prevent translation and expression of the protein. (See, e. g. , PCT International PublicationW090/l 1364, published October 4,1990 ; Sarver et al. , 1990, Science 247: 1222-1225).
  • Attenuation of a proteins activity is achieved using an agent that directly inhibits the proteins activity, such as for example an antagonist or inhibitor of the protein or an antibody specific to the protein.
  • the inhibitor may be a HSP90 inhibitor.
  • HSP90 inhibitors include geldanamycin (17-AAG), retaspimycin, and small molecule inhibitors of HSP90.
  • Other inhibitors will be well known to those skilled in the art.
  • the invention also relates to the use of a HSP90 inhibitor in the inhibition, prevention, and or treatment of invasive/metastasic cancer, especially pancreatic cancer.
  • the inhibitor is, for example, Disulfiram, 4-(N, N-dipropylamino)benzaldehyde (DPAB) or 4-(N, N- diethylamino) benzaldehyde (DEAB).
  • DPAB 4-(N, N-dipropylamino)benzaldehyde
  • DEB 4-(N, N- diethylamino) benzaldehyde
  • ALDHlAl 4-(N, N- diethylamino) benzaldehyde
  • the invention also relates to the use of a ALDHlAl inhibitor in the inhibition, prevention, and or treatment of invasive/metastasic cancer, especially pancreatic cancer.
  • a ALDHlAl inhibitor in the inhibition, prevention, and or treatment of invasive/metastasic cancer, especially pancreatic cancer.
  • this should be taken to include the administration of the protein itself, or a biologically active fragment or variant of the protein, or the administration of an agonist of the protein.
  • biologically active should be taken to mean that the fragment retains all or part of the biological functionality of the parent protein.
  • GAPDH GAPDH
  • GSN Integrin Bl
  • Integrin ⁇ 5 Integrin ⁇ 6
  • a “fragment” of a protein means a contiguous stretch of amino acid residues of at least 5 amino acids, preferably at least 6 amino acids.
  • the "fragment” will comprise at least 10, preferably at least 20, more preferably at least 30, and ideally at least 40 contiguous amino acids.
  • a "variant" of a protein shall be taken to mean proteins having amino acid sequences which are substantially identical to the wild-type protein, especially the human wild-type protein.
  • the term should be taken to include proteins or polypeptides that are altered in respect of one or more amino acid residues.
  • Such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged.
  • the variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted.
  • variants proteins which have been altered by substitution or deletion of catalytically-important residues will be excluded from the term "variant".
  • the variant will have at least 70% amino acid sequence homology, preferably at least 80% sequence homology, more preferably at least 90% sequence homology, and ideally at least 95%, 96%, 97%, 98% or 99% sequence homology with wild-type human protein.
  • sequence homology comprises both sequence identity and similarity, i.e. a polypeptide sequence that shares 70% amino acid homology with wild-type human protein is one in which any 70% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in wild-type human protein.
  • variant is also intended to include chemical derivatives of the protein, i.e. where one or more residues of the protein is chemically derivatized by reaction of a functional side group. Also included within the term variant are molecules in which naturally occurring amino acid residues are replaced with amino acid analogues.
  • Proteins and polypeptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid.
  • the proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984).
  • the invention also relates to a kit for assessing cancer status in an individual, comprising components for detecting and/or measuring the level of a protein selected from the group comprising ALDHlAl, VIM, STIPl, TPIl, KRT18, GAPDH, GSN, Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6.
  • the group comprises STIPl and ALDHlAl.
  • the kit comprises a support having an antibody specific to at least one protein selected from the above group anchored thereon.
  • the support comprises a plurality of antibodies specific to at least two proteins anchored thereon.
  • the kit comprises a support carrying a repertoire of antibodies suitable for detecting three, four, five, six, seven, eight, nine, or ten proteins anchored thereon.
  • the support is selected from the group comprising: a microtitre plate; a glass slide; a polymer membrane; and an affinity column.
  • the kit comprises an ELIS ATM kit adapted to detect one or more of the above group of proteins.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent that attenuates the activity of a protein selected from the group consisting of STIPl and ALDHlAl, and a suitable carrier or pharmaceutical excipient.
  • the agent is an oligonucelotide capable of knocking down the protein.
  • the oligonucleotide is selected from the group consisting of: siRNA; miRNA; shRNA; a ribozyme; and an antisense oligonucelotide.
  • the agent is selected from the group consisting of: an inhibitor of the protein; and an antibody that specifically binds to the protein.
  • the protein is STIPl
  • the agent is ideally is a HSP90 inhibitor.
  • the protein is ALDHlAl
  • the agent is ideally a ALDHlAl inhibitor. Details of suitable inhibitors are provided above.
  • the composition includes an effective amount of a cytotoxic agent.
  • the agent is an antibody
  • it is suitably a blocking antibody, and ideally a humanised, or fully human, antibodies.
  • Techniques for generating such antibodies are well known to the person skilled in the art.
  • the invention also relates to the use of a HSP90 inhibitor for the inhibition, prevention or treatment of an invasive/metastatic cancer.
  • the invasive/metastatic cancer is pancreatic cancer.
  • the invention also relates to methods of identifying compounds useful in the inhibition, prevention or treatment of an invasive/metastatic cancer, comprising determining a reference level of activity of a protein, contacting the protein with a candidate compound, and determining the level of activity of the contacted protein, wherein a decrease in the level of activity of the contacted protein relative to the reference level of protein activity is an indication that the candidate compound is useful in the inhibition, prevention or treatment of an invasive/metastatic cancer, wherein the protein is selected from the group consisting of: STIP 1; and ALDHlAl .
  • the protein is provided in the form of protein expressing cells, and in which the level of activity is determined by assaying for a level of expression of protein in the cells.
  • the protein is STIP
  • the STIP-expressing cells are, for example, pancreatic tumour cells.
  • the invasive metastatic cancer is pancreatic cancer.
  • the invention also relates to a method of identifying an agent that suppresses expression of STIPl or ALDHlAl protein comprising the steps of providing a source of STIPl or ALDHlAl expressing cells, treating the cells with a candidate agent, and assaying the cells for expression of STIPl or ALDHlAl, wherein a decrease in the level of expression of STIPl or ALDHlAl protein in the treated cells relative to untreated cells is an indication that the candidate agent is useful in suppressing expression of STIPl or ALDHlAl protein.
  • the invention also relates to a method of identifying an agent useful in the inhibition, prevention or treatment of an invasive/metastatic cancer, comprising a step of providing a sample of cells that express HSP90, treating the cells with a candidate agent, and assaying the cells for expression of HSP90, wherein a decrease in the level of expression of HSP90 in the treated cells relative to untreated cells is an indication that the candidate agent is useful in the inhibition, prevention or treatment of an invasive/metastatic cancer.
  • a sample of cells will be chosen that express the target protein of interest.
  • Many cancer cell lines will be useful in this regard, including (for ALDHlAl activity or espression assays), many known lung cancer cell lines.
  • the invention also relates to a method of detecting a cancer cell having an invasive/metastatic phenotype, comprising a step of assaying a biological sample from an individual for a level of a biomarker, and correlating the level with invasive/metastatic potential, wherein the biomarker is selected from the group consisting: STIPl ; and ALDHlAl.
  • the step of correlating the level with invasive/metastatic potential involves comparing the level of the protein with a reference level from a cell line having a reference invasiveness/agressiveness.
  • the biological sample is a sample of cells, and wherein the cells are stained for the biomarker, and in which an invasive/metastatic phenotype is correlated with the level of the staining of the or each of the biomarkers.
  • the Allred system may be employed in this regard, in which an Allred score of 0 correlates with no cells staining, 1 correlates with less than 1%, 2 correlates with 1% to 10%, 3 correlates with 11% to 33%, 4 correlates with 34% to 67%, and 5 correlates with more than 67%.
  • This scoring system is the sum of a proportion score and an intensity score.
  • the proportion score is an estimate of the proportion of positive cells on the entire slide and is divided into the 5 above categories.
  • the intensity score estimates the average staining intensity of positive tumor cells: 0, no staining; 1, weak positive membrane staining; 2, moderate; and 3, strong staining.
  • the 2 scores are added together to give a final numerical score ranging from 0 to 8.
  • Table 1 An alternative method in Table 1 outlines the percentage and intensity grade of staining routinely used in pancreatic cancer scoring.
  • the biological sample is a biological fluid
  • the method comprising a step of determining a level of the biomarker, and comparing the measured level of biomarker with a reference level, wherein a measured level greater than the reference level correlates with the cancer cells having an invasive/metastatic potential, and wherein a measured level less than the reference level correlates with the cancer cells not having an invasive/metastatic potential.
  • ALDHlAl aldehyde dehydrogenase
  • NAD(P)+ dependent reactions 10
  • It has been found to be approximately 9-fold up-regulated in Clone #3 compared to Clone #8, and to be more highly expressed in pancreatic tumour tissue compared to normal pancreatic tissue.
  • the amino acid sequence of ALDHlAl is provided below (SEQUENCE ID NO: 1):
  • VIM (vimentin) is a cytoskeletal protein (11). It has been found to be approximately 5.5- fold up-regulated in Clone #3 compared to Clone #8.
  • the amino acid sequence of VIM is provided below (SEQUENCE ID NO: 2):
  • Underlined sequence is minimum of four peptide identification by MALDI-TOF MS Overexpression of the protein in a cancer therefore functions as a biomarker of invasiveness/metastasis potential. Further, attenuation of expression of the protein has been shown to decrease invasiveness, and increase adhesion, in a highly invasive pancreatic cancer cell model relative to untreated cells. Thus, attenuation of VIM activity is a prophylactic or therapeutic treatment for cancer invasiveness/metastasis.
  • STIPl stress induced phosphoprotein 1
  • Hsp70 and Hsp90 mediates the association of the molecular chaperones Hsp70 and Hsp90 (12). It has been found to be approximately 2.6-fold up- regulated in Clone #3 compared to Clone #8.
  • the amino acid sequence of STIPl is provided below (SEQUENCE ID NO: 3): MEQVNELKEKGNKALSVGNIDDALQCYSEAIKLDPHNHVLYSNRSAAYAK KGDYOKAYEDGCKTVDLKPDWGKGYSRKAAALEFLNRFEEAKRTYEEGLK HEANNPQLKEGLQNMEARLAERKFMNPFNMPNLYQKLESDPRTRTLLSDP TYRELIEOLRNKPSDLGTKLODPRIMTTLSVLLGVDLGSMDEEEEIATPP PPPPPKKETKPEPMEEDLPENKKOALKEKELGNDAYKKKDFDTALKHYDK AKELDPTNMTYITNQAA VYFEKGDYNKCRELCEKA
  • Overexpression of the protein in a cancer therefore functions as a biomarker of invasiveness/metastasis potential.
  • attenuation of expression, or activity, of the protein has been shown to decrease invasiveness, and increase adhesion, in a highly invasive pancreatic cancer cell model relative to untreated cells, and in other cell lines of pancreatic cancer.
  • attenuation of STIPl activity is a prophylactic or therapeutic treatment for cancer invasiveness/metastasis, especially in pancreatic cancer.
  • TPIl triphosphate isomerise a
  • the amino acid sequence of TPIl is provided below (SEQUENCE ID NO: 4):
  • Underlined sequence is minimum of four peptide identification by MALDI-TOF MS Overexpression of the protein in a cancer therefore functions as a biomarker of invasiveness/metastasis potential.
  • Cytoskeletal protein KRTl 8 (keratin 18) has been found to be approximately 3-fold down-regulated in Clone #3 compared to Clone #8.
  • the amino acid sequence of KRTl 8 is provided below (SEQUENCE ID NO: 5):
  • Underexpression of the protein in a cancer therefore functions as a biomarker of invasiveness/metastasis potential.
  • Glycolytic protein GAPDH (glyceraldehyde 3-phosphate dehydrogenase) has been found to be approximately 2.6-fold down-regulated in Clone #3 compared to Clone #8.
  • the amino acid and nucleic acid sequence of GAPDH is provided below (SEQUENCE ID
  • IALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE Underexpression of the protein in a cancer therefore functions as a biomarker of invasiveness/metastasis potential .
  • GSN gelsolin
  • Isoform b of the protein has been found to be approximately 21 -fold down-regulated in Clone #3 compared to Clone #8.
  • the amino acid sequence of GSN isoform b is provided below (SEQUENCE ID No: 7):
  • Underexpression of the protein in a cancer therefore functions as a biomarker of invasiveness/metastasis potential. Further, attenuation of expression of the cytoplasmic form of the protein has been shown to increase invasiveness in a pancreatic cancer cell model of low invasiveness relative to untreated cells.
  • Integrin Bl, Integrin ⁇ 5, and Integrin ⁇ 6 are cell surface receptors known to be associated with receptors of fibronectin and laminin.
  • the proteins are overexpressed in a low invasiveness cell line compared with normal and high invasiveness cell models of pancreatic cancer. Attenuation of the protein in a low invasiveness cell model of pancreatic cancer by siRNA has been shown to increase the invasiveness of the treated cells.
  • B. Adhesion of MiaPaCa-2, Clone #3 and Clone #8 to ECM proteins: matrigel, laminin, fibronectin, collagen type IV and type I. Results are expressed as absorbance at 405 nm with a reference wavelength of 620 nm. Data shown is mean ⁇ standard deviation (n 3). Student's t-test; p ⁇ 0.05*, 0.01**, 0.005*** .
  • Figure 3 Bar graph displays the total number of cells invading under control conditions and also after 24 hr incubation on matrigel, scatter graph displays the total number of superinvading cells counted after 24 hrs incubation on matrigel of Clone #8, MiaPaCa-2 and Clone #3.
  • B Images showing the morphology of (i) MiaPaCa-2, (ii) Clone #3 and (iii) Clone #8. Magnification, 20Ox, scale bar, 200 ⁇ m
  • Figure 4 2D DIGE expression map of Cy2, Cy3 and Cy5 labelled Clone #8 compared to Clone #3 proteins.
  • Figure 5 Immunofluorescence, 3D spot images and Western blot images of A vimentin and B cytokeratin 18 protein expression in MiaPaCa-2, Clone #3 and Clone #8.
  • Figure 6 3D spot images and Western blot images of A. ALDHlAl, B. STIPl C. TPIl and D. GAPDH expression in MiaPaCa-2, Clone #3 and Clone #8. BiP used as loading control.
  • FIG. 7 Western blot of A. Integrin ⁇ l B. Integrin ⁇ 2 C. Integrin ⁇ 5 D. Integrin ⁇ 6 and ⁇ -actin (below) used as loading control in (1) MiaPaCa-2, (2) Clone #3 and (3) Clone #8.
  • Figure 8 A. Invasion of Clone #8 through matrigel, laminin and fibronectin and motility assay. B. Adhesion assay of Clone #8 to matrigel, laminin and fibronectin. C. Anoikis assay. Experiments were performed 48 hours post-transfection with two different exon targeted siRNA integrin Beta 1. Untransfected- and scrambled siRNA transfected- cell lines were the controls for this experiment. Student's t-test;p ⁇ 0.05*, 0.01 **, 0.005*** .
  • Figure 9 A. Invasion through matrigel, laminin and fibronectin.
  • Figure 10 Western blot of siRNA ALDHlAl knockdown in Clone #3. Three independent target siRNA of ALDHl Al were transfected into Clone #3 cells. Protein was harvested 48 hrs post-transfection and used to determine an ALDHl Al- siRNA specific decrease at protein level in response to siRNA transfection by Western blot, ⁇ -tubulin antibody was used to demonstrate even loading between the samples.
  • Figure 1 1 (A) Invasion assays of Clone #3 (i) under control conditions (ii) transfected with scrambled siRNA (iii) transfected with ALDHlAl siRNA (1) (iv) transfected with ALDHlAl siRNA (2) (v) transfected with ALDHlAl siRNA (3), 48 hrs post transfection. Magnification, 20Ox. Scale bar, 200 ⁇ m. (B) Total number of Clone #3 cells invading post ALDHlAl siRNA transfection. Statistics: p ⁇ 0.05*, 0.01 **, 0.005*** (unpaired t-test) to scrambled controls
  • Figure 12 Percentage adhesion of Clone #3 untreated, scrambled and treated with three target ALDHlAl siRNAs to matrigel 48 hrs after transfection. Results are expressed as % adhesion relative to untreated control cells. Data shown is mean ⁇ standard deviation
  • Figure 14 Western blot analysis of ALDHlAl cDNA transient transfection in Clone #8. Two time points of 48 hrs and 72 hrs post cDNA transfection were used and ⁇ -actin was used as loading control.
  • Figure 15 Invasion assays of (A) (i) Clone #8 under control conditions (ii) Clone #8 transfected with empty vector (EV) (iii) Clone #8 transfected with ALDHlAl cDNA. Magnification, 20Ox. Scale bar, 200 ⁇ m. (B) Invasion assay of Clone #8 of total number of cells invading 48 hrs post ALDHlAl cDNA transfection. Statistics: p ⁇ 0.05*, 0.01 **, 0.005*** (unpaired t-test) to empty vector control
  • Figure 17 Western blot of Clone #3 control, treated with 5 ⁇ M ATRA for 48 hrs and after continuous ATRA treatment, ⁇ -actin was used as loading control.
  • Figure 18 (A) Invasion of (i) Clone #3 and (ii) Clone #8 after 8 days continuous exposure to 5 ⁇ M ATRA. (B) Total number of cells invading. Statistics: p ⁇ 0.05*, 0.01**, 0.005*** (unpaired t-test) to control Figure 19: Morphology of Clone #3 (i) under normal culture conditions (ii) 5 ⁇ M ATRA treatment (iii) Clone #8 under normal culture conditioned and (iv) 5 ⁇ M ATRA treatment. Magnification, 10Ox. Scale bar, 200 ⁇ m.
  • Figure 20 Western blot of siRNA VIM knockdown in Clone #3. Three independent target siRNA of VIM were transfected into Clone #3 cells.
  • Protein was harvested 48 hrs post-transfection and used to determine a VIM-siRNA specific decrease at protein level in response to siRNA transfection by Western blot, ⁇ - tubulin antibody was used to demonstrate even loading between the samples. This is a representative picture of at least 3 independent analyses.
  • Figure 21 (A) Invasion assays of Clone #3 (i) under control conditions (ii) transfected with scrambled siRNA (iii) transfected with VIM siRNA (1) (iv) transfected with VIM siRNA (2) (v) transfected with VIM siRNA (3). Magnification, 20Ox. Scale bar, 200 ⁇ m. (B) Invasion assay of Clone #3 of total number of cells invading post siRNA vimentin transfection. Statistics: p ⁇ 0.05*, 0.01**, 0.005*** (unpaired t-test) to scrambled control
  • Figure 25 Morphology of (i) MiaPaCa-2, (ii) Clone #3 and (iii) Clone #8 and after transfection (iv) Clone #3 control (v) Clone #3 scrambled (vi) Clone #3 siRNA kinesin (vii) Clone #3 transfected with VIM (1) (viii) Clone #3 transfected with VIM (2) and (ix) Clone #3 transfected with VIM (3) 48 hours post-transfection. Magnification at 2Ox, scale bar, 200 ⁇ m.
  • Figure 26 Western blot of siRNA STIPl knockdown in Clone #3. Three independent target siRNA of STIPl were transfected into Clone #3 cells.
  • Protein was harvested 48 hrs post-transfection and used to determine a STIPl -siRNA specific decrease at protein level, ⁇ -tubulin antibody was used to demonstrate even loading between the samples. This is a representative picture of at least 2 independent analyses.
  • Figure 27 (A) Invasion assays of Clone #3 (i) under control conditions (ii) transfected with scrambled siRNA (iii) transfected with STIPl siRNA (1) (iv) transfected with STIPl siRNA (2) (v) transfected with STIPl siRNA (3). Magnification, 20Ox. Scale bar,
  • Figure 31 Western blot validation and 3D spot images of A. GSN, B. NDPK, C. LGALSl, D. ALDHlAl, E. BiP (loading control) in CM#3 and CM#8.
  • Figure 32 A Western blot of two independent target siRNA-GSN knockdown in CM#8. Bip antibody was used to demonstrate even loading between the samples.
  • Figure 33 A. Western blot of GSN expression in MiaPaCa-2, Clone #3 and Clone #8 cells and after 24 hrs grown on matrigel. ⁇ -actin, used as loading control.
  • Figure 34 A. Western blot of ALDHlAl knockdown in CM#3 untreated control, scrambled, siRNA ALDHlAl (1), siRNA ALDHlAl (2) and siRNA ALDHlAl (3).
  • Figure 35 A. Proteomics analysis (2D DIGE MALDI-TOF MS) of STIPl up-regulation in Clone #3 compared to Clone #8 as shown by 3D spot image and protein expression map (PEM).
  • Figure 36 Immunoblotting for STIPl, HSP70, HSP90, AbI, HER2 and AKT after transfection with STIP 1 -siRNA in BxPc-3, Panc-1 and Clone #3.
  • Figure 37 Invasion assays of (A.) Panc-1 (B.) BxPc-3 48 hrs post transfection with scrambled siRNA and three independent siRNA sequences against STIPl. The total number of invading cells was determined by counting the number of cells per field in 10 random fields, at 200* magnification. The average number of cells per field was then multiplied by a factor of 140 (growth area of membrane/field area viewed at 200* magnification (calibrated using a microscope graticule)).
  • FIG 38 Immunoblotting for MMP2 after transfection with STIPl-siRNA in, Panc-1 and BxPc-3.
  • Figure 39 (A.) Invasion assay picture representations of invasion under control conditions and 24 hr treatment with 17 AAG in Panc-1 and BxPc-3 cells. Invasion is significantly reduced. (B.) Percentage survival of panel of 8 pancreatic cancer cell lines treated with 17 AAG chemosensitivity assay.
  • Figure 40 IHC detection of STIPl (A-D) in pancreatic cancer and normal pancreas tissues.
  • A Strong STIPl cytoplasmic staining in PC tumour ducts.
  • B-C Strong STIPl expression in poorly differentiated PC tumours.
  • D Moderate staining of normal pancreas ducts and acinar cells.
  • FIG 41 Toxicity profiles of pancreatic cancer cell lines (MiaPaCa-2, Panc-1, BxPc-3, Clone#3 and Clone #8), breast cancer cell lines (SKBR3 and T47D) and lung cancer cell line (DLKP) to disulfiram.
  • Figure 42 IHC detection of ALDHl Al (D-H) in pancreatic cancer and normal pancreas tissues.
  • D Moderate staining of normal pancreas ducts and acinar cells.
  • E ALDHlAl highly expressed in well differentiated PC tumour.
  • F-G Weak ALDHlAl staining observed in ⁇ 10% of poorly differentiated PC tumours.
  • H Positive staining in epithelial cells of normal pancreas.
  • the human pancreatic cell line MiaPaCa-2 was obtained from the European Collection of
  • Matrigel (Sigma-Aldrich, UK) was coated onto flasks (1 ml/25 cm 2 ) at a concentration of 1 mg/ml. The coated flasks were then placed at 4 0 C overnight. The flasks were placed into an incubator at 37 0 C for approximately 2 hrs to allow the matrigel polymerise. The excess media in the flasks was then removed and fresh complete media containing the cell suspension was added. Cells attached to the matrigel on the bottom of the flask and after 24 hrs were removed with 0.5 ml/T25 cm 2 dispase (BD Biosciences). Dispase is a bacillus derived neutral metaloprotease that recovers cells cultured on matrigel.
  • pancreatic cancer cell line-conditioned media Clone #3 and Clone #8 monolayers were cultured in Tl 75 cm 3 flasks until approximately 60% confluent in culture medium. Cells were then washed X3 with serum free (SF) DMEM and incubated for 1 hr with SF DMEM. Cells were washed X3 again in SF DMEM, then placed in SF DMEM for 72 hrs. At the time of collection, cellular debris was removed by centrifugation and filtration through 0.22 ⁇ m filter; aliquots were frozen at -80 °C until analysed. Invasion assays Invasion assays were performed using an adapted method (14).
  • Matrigel was diluted to 1 mg/ml in serum free DMEM. 100 ⁇ l of matrigel was placed into each insert (Falcon) (8.0 ⁇ m pore size) in a 24 well plate (Costar). The coated inserts were incubated overnight at 4 0 C. The following day, the matrigel was allowed polymerise at 37 0 C for 1 hr. The inserts were then washed with DMEM, 100 ⁇ l of lxl0 5 /100 ⁇ l cells in complete DMEM and 100 ⁇ l of CM supplemented with 5 % serum was added onto the insert. 250 ⁇ l of total DMEM: 250 ⁇ l CM supplemented with 5% serum was added to the 24-well.
  • CM#3 and CM#8 Three 50 ml of CM#3 and CM#8 (3 biological replicates and two technical replicates/CM of cell line) were concentrated using 10,000 molecular weight cut-off (Millipore); samples were cleaned-up using ready-prep 2D clean-up kit (BioRad). Protein concentration was determined using the BSA protein assay kit (Bio-Rad). CM samples were labelled with N-hydroxy succinimidyl ester-derivatives of the cyanine dyes Cy2, Cy3 and Cy5 (15). Typically, 50 ⁇ g of the CM was minimally labelled with 200 pmol of either Cy3 or Cy5 for comparison on the same 2-D gel.
  • Immobilised 24 cm linear pH gradient (IPG) strips pH 3-11, were rehydrated in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer, 50 mM DTT) overnight, according to manufactures guidelines.
  • IEF was performed using as IPGphor apparatus (GE Healthcare) for 40 kV/h at 20 0 C with resistance set at 50 mA. Strips were equilibrated for 20 min in 50 mM Tris-HCL, pH 8.8, 6 M urea, 30% v/v glycerol, 1% w/v SDS containing 65 mM DTT and the for 20 min in the same buffer containing 240 mM iodoacetamide.
  • Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). 35 ⁇ g of protein was separated by 7.5% and 15% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membrane, efficiency and equal loading of protein was evaluated by Ponceau S staining. Membranes were blocked at 4 0 C overnight in TBS (25mM Tris-HCl, pH 7.4, 15OmM NaCl, 2.7mM KCl) containing 5% (w/v) lowfat milk powder.
  • TBS 25mM Tris-HCl, pH 7.4, 15OmM NaCl, 2.7mM KCl
  • Membranes were probed with monoclonal antibodies, anti-gelsolin (Sigma), anti-nucleotide diphosphate kinase (Abeam), anti-galectin-1 (Abeam) and anti- aldehyde dehydrogenase (Calbiochem) anti-vimentin (Sigma), anti-stress-induced phosphoprotein 1 (Santa Cruz), anti-triosephosphate isomerase (Abeam), anti-GAPDH (Ambion), anti-cytokeratin 18 (Santa Cruz).
  • Integrin anti- ⁇ l, anti- ⁇ 5 and anti- ⁇ 6 monoclonal antibodies were obtained from Becton Dickinson (BD Biosciences UK) and Chemicon (Europe, UK) respectively. Secondary antibodies, anti-mouse and anti-rabbit were obtained from Sigma. Protein bands were detected with Luminol reagent (Santa Cruz Biotechnology).
  • Cells were transfected with two different GSN siRNA targets (Ambion, #8127, 8031) and three different ALDHlAl siRNA targets (Ambion, #106197, #106196, and #106195) and scrambled siRNA (Ambion, # 17010), using NeoFx transfection reagent (Ambion, AM4511), according to the manufacturer's instructions. Briefly, 3 x 10 5 pancreatic cancer cells were seeded in six-well plates in complete medium at 70% confluence and after 24 hrs transfected with the indicated siRNA, scrambled siRNA and control. After 32 hrs, the media was removed, washed 3x in SF DMEM and 1 ml of SF DMEM was added onto the cells.
  • SiRNA transfected SF CM was collected, centrifuged and filtered through a 0.22 ⁇ m filter.
  • SiRNA transfected SF CM was concentrated using 10,000 molecular weight cut-off concentrators (Millipore); samples were cleaned-up using ready-prep 2D clean-up kit (BioRad) and protein concentration was determined using the BSA protein assay kit (Bio- Rad). Western blot analysis was then carried out to assess efficient transfection. All experiments were repeated in triplicate.
  • siRNA experiments in 6- well plates were set up using 2 ⁇ l NeoFx to transfect 30 nM siRNA in a cell density of 3x10 5 per well of a 6-well plate.
  • Transfection medium, optimem (Ambion) was removed after 24 hours and replaced with fresh growth medium.
  • the transfected cells were collected for Western blot and assayed for changes in invasion capacity at 48 hours using the in vitro invasion assay.
  • the target cell line was trypsinised in 6-well plates and set up at 50-70% confluency. Following incubation overnight at 37 0 C, transfection mixtures of 5 ⁇ l transfection reagent (lipofectamine) in 125 ⁇ l optimem (Ambion) were prepared and combined with 2 ⁇ g cDNA in 125 ⁇ l optimem and incubated for 15 minutes at RT. Control wells were untreated and empty vector controls contained transfection mixture without cDNA in the presence of empty plasmid, pCMV6-XL5 (Origene). The transfection mixture was added directly onto cells in 6-well plate and incubated for a further 48 hrs.
  • Integrins Two integrin ⁇ l (ITGBl) target siRNAs (#109877, #109878 (validated) Ambion Inc.) were used to silence integrin ⁇ l expression.
  • Three integrin ⁇ 5 (ITGA5) target siRNAs (#106728, #1 1 1 1 13, #106729 Ambion Inc.) and two integrin cc6 (ITGA6) target siRNAs (#8146, #103827 (validated) Ambion Inc.) were used to silence the respective target genes.
  • Solutions of siRNA at a final concentration of 30 nM were prepared in OptiMEM (GibcoTM).
  • NeoFX solution was prepared in OptiMEM and incubated at RT for 10 minutes.
  • neoFX solution was added to each siRNA solution, mixed well and incubated for a further 10 minutes.
  • 100 ⁇ l of neoFX/OptiMEM solutions were added into a 6 well plate in duplicate.
  • Mia clone #8 (3 x 10 5 ) cells were added onto the siRNA solution. The plates were gently mixed and incubated for 24 hours. The transfection mixture was removed and replaced with fresh medium. Positive control kinesin (Ambion Inc.) was included in each triplicate experiment. Invasion, adhesion and anoikis assays were then carried out 48 hours after transfection, as previously described.
  • Invasion assays 100 ⁇ l of matrigel (1 mg/ml) was placed into each invasion insert (Falcon) (8.0 ⁇ m pore size) in a 24 well plate (Costar). The coated inserts were incubated overnight at 4 0 C. Matrigel was allowed polymerize at 37 0 C for 1 hr, then washed with serum-free DMEM. 100 ⁇ l of fresh DMEM containing 5% serum was added to the wells and lxl0 5 /100 ⁇ l cells were seeded onto the insert. 500 ⁇ l of fresh DMEM with 5% serum was added to the well. After 24 hour incubation, the inside of the insert was wiped with a wet cotton swab.
  • the under surface was gently rinsed with PBS and stained with 0.25% crystal violet for 10 minutes, rinsed again with sterile water and allowed to dry.
  • the inserts were then viewed under the microscope and the number of cells per field in 10 random fields were counted at 200 ⁇ magnification.
  • the average number of cells per field was then multiplied by a factor of 140 (growth area of membrane/field area viewed at 200 ⁇ magnification (calibrated using a microscope graticule)). The mean values were obtained from a minimum of three individual experiments and were subjected to /-tests.
  • siRNA experiments were set up using 2 ⁇ l NeoFx (Ambion, AM4511), to transfect 30 nM siRNA at a cell density of 3x10 5 /well/ml of a 6-well plate.
  • Immunoblotting Whole protein was extracted from cell lysates using Ix lysis buffer (50 mM Tris-Cl, 150 mM NaCl, and 0.5% NP-40). Lysates were centrifuged for 10 min at 14,000 rpm at 4° C. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). 35 ⁇ g of protein was separated by 7.5% and 15% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membrane, efficiency and equal loading of protein was visualised by Ponceau S staining.
  • Membranes were blocked at 4 0 C overnight in TBS (25mM Tris-HCl, pH 7.4, 15OmM NaCl, 2.7mM KCl) containing 5% (w/v) low fat milk powder.
  • Membranes were probed with monoclonal antibodies, anti-aldehyde dehydrogenase (Abeam), anti-stress-induced phosphoprotein 1 (Santa Cruz) (Abeam), HSP90 (Cell signalling), HSP70 AND AbI (Abeam), HER2, AKT (Calbiochem) and ⁇ - actin (Sigma-Aldrich) (loading control).
  • Secondary antibodies, anti-mouse, anti-rabbit and anti-goat were obtained from Sigma. Protein bands were detected with Luminol reagent (Santa Cruz Biotechnology).
  • IHC Analysis Patients The patient group consisted of 5 consenting patients diagnosed with primary tumours of the pancreas. All patients were treated at St. Vincent's University Hospital (SVUH), Dublin in 2005. IHC studies on tumour-free pancreatic tissue were performed using corresponding non-cancerous tissue. Pathological material was examined on each case by SK. Formalin-fixed paraffin-embedded pancreatic tumour tissue and corresponding normal pancreas was available for all patients. Representative 4- ⁇ m sections of tissue block were cut using a microtome, mounted onto poly-1-lysine coated slides and dried overnight at 37 0 C. Slides were stored at room temperature until required.
  • the slides were immunohistochemically stained using primary antibodies specific for ALDHlAl and STIPl from Abeam.
  • the staining procedure includes an antigen retrieval step consisting of 20-minute incubation in pH 9.0 buffer (TARGET Retrieval, Dako) in a 95°C water bath followed by cooling to room temperature. Staining was performed using an automated staining apparatus for IHC (Autostainer, Dako) according to the manufacturer's guidelines. The slides were counterstained with haematoxylin.
  • siRNA scrambled transfected cells were used as control compared to siRNA treated samples. This was to ensure no 'off-target' effects of the transfection procedure.
  • Non-treated controls were used to ensure scrambled siRNA was having no effects and to normalise data.
  • a p value of ⁇ 0.05 * was deemed significant, p value ⁇ 0.01 ** was deemed more significant, p value ⁇ 0.005 *** was deemed highly significant.
  • FIG. 1 A illustrates the invasion of MiaPaCa-2 and sub-clones Clone #3 and Clone #8 through matrigel, laminin, fibronectin and collagens type IV and I.
  • TPIl was also investigated by Western blot (Fig 6 C); however, the results were not consistent with the 2D-DIGE analysis. This may be due to a number of reasons, possibly due to the presence of multiple isoforms and variants of TPIl.
  • GAPDH is also higher in the less invasive cell line, Clone #8 compared to Clone #3 or the parental cell line, MiaPaCa-2. Expression of GAPDH decreases as the invasion status of the cells increases.
  • Integrin expression Expression of integrins ⁇ l, ⁇ 2, ⁇ 5 and ⁇ 6, which are associated with adhesion to laminin and fibronectin were examined in the cell lines, by western blotting ( Figure 7 A-D). Compared to MiaPaCa-2, Clone #8 showed a higher expression of integrins ⁇ l, ⁇ 2, ⁇ 5 and integrin ⁇ 6. Expression of integrins ⁇ l, ⁇ 2, ⁇ 5 and ⁇ 6 were lower in Clone #3. Integrin ⁇ l knockdown:
  • Integrin ⁇ 5 and ⁇ 6 expression was assessed in Figure 7 (C-D) in the model of pancreatic cancer cell lines.
  • siRNA experiments targeting these integrins were also carried out in Clone #8 cells.
  • Expression of integrin ⁇ 5 and ⁇ 6 was reduced ( Figure 7 F-G).
  • Aldehyde dehydrogenase was identified as a protein potentially involved in invasion in our in vitro pancreatic cancer cell line model. The analysis showed that ALDHlAl was 9-fold up-regulated in the more invasive sub-population, Clone #3 compared to the low invasive cell line, Clone #8 (Table 1).
  • the protein aldehyde dehydrogenase IAl was shown to be up-regulated as the invasion status of the cell lines increased, therefore the more invasive cell line, Clone #3 expressed an abundance of the protein. Clone #3 was used for siRNA knockdown and further functional analysis.
  • Figure 10 shows by Western blot, the efficient knockdown of ALDHlAl in three siRNA treated Clone #3 cells compared to non-treated control and siRNA scrambled transfected cells.
  • Adhesion assays were also carried out to assess the involvement of ALDHlAl in adhesion to matrigel.
  • anoikis assays were performed with ALDHlAl siRNAs.
  • Figure 13 shows that anoikis is modestly induced in siRNA ALDHlAl (1) and (2) transfected cells compared to scrambled treated cells.
  • no significant difference is observed in anoikis of cells transfected with ALDHlAl siRNA (1) and (2) compared to the scrambled controls.
  • Proliferation assays were carried out over 5 days after transfection of ALDHlAl siRNAs into Clone #3 cells.
  • Figure 14 displays the percentage survival of transfected cells relative to untreated control. Kinesin was used as a control for efficient transfection. There was no significant difference in proliferation of siRNA ALDHlAl treated cells compared to control cells, therefore loss of ALDHlAl did not affect proliferation in Clone #3 cells.
  • Western blot validation confirmed that ALDHlAl expression was highest in the invasive sub-population, Clone #3 and lowest in the lesser invasive sub- population, Clone #8.
  • FIG. 16 shows representative pictures of invading cells, and (B) highlights the total number of invading cells of Clone #8 control (untreated), Clone #8 transfected with empty vector and Clone #8 transfected with ALDHlAl cDNA.
  • Chemosensitivity assays were carried out in the three cell lines, MiaPaCa-2, Clone #3 and
  • Clone #8 4-hydroxycyclophosphamide (4-HC) and mafosfamide are known to be detoxified by ALDHlAl .
  • 4-diethylaminobenzaldehyde (DEAB) is a specific inhibitor of ALDHlAl and ALDHlAl converts retinal to retinoic acid.
  • the high invasive cell line, Clone #3 is more resistant to the cytotoxic effects of cyclophosphamide metabolite, 4-HC and analogue, mafosfamide.
  • the parental cell line, MiaPaCa-2 and the low invasive cell line, Clone #8 are more sensitive to the drugs (Table 4).
  • IC 50 S calculated represent half maximal inhibitory concentration of each drug in MiaPaCa-2, Clone #3 and Clone #8.
  • Chemosensitivity assays were performed on Clone #3 cells transfected with ALDHlAl siRNA to determine whether ALDHlAl silencing sensitised the cells to the toxic effects of 4-HC.
  • Figure 17 shows that ALDHlAl siRNA slightly increased sensitivity to 4-HC and may be associated with 4-HC resistance in pancreatic cancer cells.
  • Table 5 outlines the IC 5 oS of Clone #3 untreated control, scrambled and transfected with three independent siRNA ALDHlAl .
  • ALDHlAl acts as a catalyst irreversibly converting retinaldehyde to retinoic acid (RA). Analyses were performed to investigate whether accumulation of intracellular retinoic acid may lead to the suppression of ALDH expression (Moreb et al., 2005). Clone #3 and Clone #8 were incubated with 1.5 ⁇ g/ml (5 ⁇ M) of ATRA to assess a possible feedback loop of high levels of retinoic acid on ALDHlAl expression. This effect was measured by Western blot, invasion assays and morphological changes in the cells.
  • ALDHlAl expression was determined by Western blot in Clone #3 cells treated with 5 ⁇ M ATRA for 48 hrs and continuous 5 ⁇ M ATRA, treatment.
  • Figure 18 shows by Western blot that ALDHlAl expression is not altered in Clone #3 after ATRA treatment.
  • Figure 20 (i) displays the morphology of control Clone-#3 cells, while (ii) highlights the morphology of these cells after continuous exposure to ATRA. These results show that the morphology is unchanged.
  • Disulfiram an inhibitor of ALDHlAl, decreases invasion and is more toxic to pancreatic cancer cell lines compared to ALDHlAl expressing breast (SKBR3 and T47D) and lung cancer (DLKP) cell lines (Figure 41).
  • ALDHlAl as a marker for stem cells.
  • Vimentin was identified as a protein involved in invasion in our in vitro pancreatic cancer cell line model. The analysis showed that VIM was 5.5-fold up-regulated in the more invasive sub-clone, Clone #3 compared to the low invasive cell line, Clone #8 (Table 1).
  • the protein vimentin was shown to be up-regulated as the invasion status of the cell lines increased, therefore the more invasive cell line, Clone #3 expressed an high levels of the protein. Clone #3 was used for siRNA knockdown and further functional analysis.
  • Figure 21 shows by Western blot the efficient knockdown of VIM in Clone #3 cells transfected with three VIM siRNAs compared to non-treated control and siRNA scrambled transfected cells.
  • the loss of VIM expression through siRNA knockdown in Clone #3 cells reduces invasion and confirms proteomics results.
  • Adhesion assays were also carried out to assess the involvement of vimentin in adhesion of Clone #3 to matrigel.
  • Figure 23 shows the % adhesion relative to untreated control cells.
  • FIG. 25 displays the percentage survival of Clone #3 cells transfected with VIM siRNAs. Loss of VIM did not affect proliferation in Clone #3 cells, and VIM is not essential for proliferation of these cells. Effect of VIM siRNA on epithelial to mesenchymal transition (EMT) in Clone #3 cells EMT is characterised by morphological and behavioural changes in cells (Maeda et al., 2005). Investigations into the involvement of VIM, a mesenchymal marker (Leader et al., 1987) were determined.
  • EMT epithelial to mesenchymal transition
  • Figure 26 (i-iii) shows the morphological changes of Clone #3 and Clone #8 compared to the parental cell line, MiaPaCa-2 under normal culture conditions.
  • Clone #3 exhibits a more fibroblast phenotype with spindle shaped elongated cells.
  • Clone #8 cells appear rounded and grow in clusters.
  • Clone #3 transfected with siRNA VIM exhibits a rounded phenotype as observed in Clone #8 cells.
  • the morphological changes of Clone #3 after loss of VIM expression may implicate the role of vimentin in the epithelial to mesenchymal transition.
  • STIPl was identified as a protein 2-fold up-regulated in the high invasive sub-population Clone #3, compared to the low invasive sub-population, Clone #8 (Table 2).
  • the expression of STIPl increased as the invasion status of the cells increased suggesting it may correlate to invasion in pancreatic cancer.
  • the protein stress induced phosphoprotein 1 was shown to be up-regulated as the invasion status of the cell lines increased, therefore the more invasive cell line, Clone #3 expressed high levels of the protein. Clone #3 was used for siRNA knockdown and further functional analysis.
  • Figure 27 showed by Western blot the efficient knockdown of STIPl in siRNA treated Clone #3 cells compared to non-treated and scrambled controls.
  • FIG. 28 displays (A) representative pictures of the level of invasion and (B) the total number of cells invading.
  • STIPl is associated with invasive pancreatic carcinoma STIPl is identified by 2D DIGE followed by MALDI-TOF MS as 2-fold up-regulated in Clone #3, a highly invasive sub-population of the human pancreatic cancer cell line MiaPaCa-2, compared to the low invasive Clone #8 (Fig 35 A). STIPl expression in a panel of pancreatic cancer cell lines was also examined. Expression of STIPl corresponded with the invasive status of the cell lines (Fig 35 B).
  • STIPl-siRNA was used to knock down STIPl expression in three invasive human pancreatic cancer cell lines: BxPc-3, Panc-1 and Clone #3. 48 hours post transfection, STIPl expression was reduced in BxPc-3, Panc-1 and Clone #3 (Fig 36).
  • STIPl siRNA did not alter levels of HSP70 or HSP90 expression in BxPc-3, Panc-1 cell lines.
  • the expression of the HSP90 client proteins, HER2 and AbI were significantly reduced with STIPl-siRNA transfection.
  • AKT expression was not altered after STIPl- siRNA transfection (Fig 36)
  • FIG. 5 A highlights the successful knockdown of GSN secretion in CM#8 by two independent siRNA targets relative to siRNA scrambled and controls (untreated) CM#8.
  • FIG. 6 A shows by Western blot that cytoplasmic GSN is expressed only in the lysates of Clone #8 cells, however after 24 hrs incubation on matrigel (simulating the in vitro invasion assay) the cytoplasmic GSN is further enhanced after cell-matrigel contact.
  • FIG. 7 A shows the efficient knock down of ALDHlAl in CM#3 by three independent targets by Western Blot. ALDHl Al -siRNA treated CM#3 was added into the invasion assay of Clone #3.
  • Figure 7 B highlights representative photographs of invasion inserts and the total number of invading cells, whereby reduction of ALDHlAl expression resulted in a significant decrease in invasive abilities of Clone #3 cells.

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Abstract

L'invention porte sur un procédé pour l'inhibition, la prévention ou le traitement d'un cancer invasif/métastatique dans un individu en ayant besoin, lequel procédé comprend une étape de traitement de l'individu par un agent capable d'atténuer l'activité d'une protéine choisie dans le groupe constitué par : STIPl; et ALDHlAl. De manière appropriée, le cancer invasif/métastatique est un cancer pancréatique, typiquement des métastases sélectionnées dans le groupe consistant en : métastases osseuses; métastases du poumon; métastases du foie; métastases de la moelle osseuse; métastases du sein; et métastases du cerveau. L'agent est un agent qui supprime l'expression de la protéine, par exemple, l'ARNsi, l'ARNmi, l'ARNsh, un ribozyme ou un oligonucléotide antisens.
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CN112083171A (zh) * 2020-09-07 2020-12-15 中国人民解放军总医院第八医学中心 Ku70蛋白T455位点磷酸化抑制剂及其应用

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