WO2009121924A1 - Marqueurs métastatiques - Google Patents

Marqueurs métastatiques Download PDF

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WO2009121924A1
WO2009121924A1 PCT/EP2009/053923 EP2009053923W WO2009121924A1 WO 2009121924 A1 WO2009121924 A1 WO 2009121924A1 EP 2009053923 W EP2009053923 W EP 2009053923W WO 2009121924 A1 WO2009121924 A1 WO 2009121924A1
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protein
cancer
expression
lung cancer
proteins
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PCT/EP2009/053923
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English (en)
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Stephen Fey
Peter Mose Larsen
Kelan Zhang
Wrzesinski Krzystof
Peter Roepstorff
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Drugmode Aps
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the invention relates to the identification and use of a protein or gene expression profile with clinical relevance to cancer metastasis.
  • the invention provides the identities of proteins that are correlated with lung cancer metastasis.
  • the protein expression profiles may be used to predict the survival of subjects afflicted with cancer and to predict cancer recurrence.
  • the profiles may also be used in the study and/or diagnosis of cancer cells and tissue, including the grading of invasive lung cancer, as well as for the study and/or determination of prognosis of a patient.
  • the profiles are used to determine the treatment of cancer based upon the likelihood of life expectancy and metastasis.
  • Tumors can be either benign or malignant. Benign tumors are not cancerous; they do not spread to other parts of the body, and are not a threat to life. They can usually be surgically removed, and in most cases, do not come back. Malignant tumors are cancerous, and can invade and damage nearby tissues and organs. Malignant tumor cells may metastasize, entering the bloodstream or lymphatic system.
  • Cancer metastasis represents the major cause of morbidity and death for cancer patients.
  • Metastasis is a complex and sequential process including invasion of tumor cells into adjacent tissues, intravasation, transport through the circulatory system, arrest at a secondary site, extravasation and growth in a secondary organ.
  • the genetic basis of tumorigenesis can vary greatly whereas the basic steps required for metastasis are similar for all tumor cells.
  • the present invention is based on the differential expression of the proteins of supplementary Tables 1-3. So far no prior art disclosure has envisaged that this protein profile is useful for the prediction of risk of metastasis.
  • Adenocarcinoma is the most prevalent type of non-small-cell lung cancer (NSCLC) representing 80% of all human lung cancers [The lung in Pathologic basis of diseases,
  • the present inventors have developed a simple procedure to predict the risk for cancer metastasis, and particularly lung cancer metastasis, which procedure is based on the differential expression of specific proteins.
  • the fact that several of the proteins identified have been associated with other cancer types is strongly suggestive of the fact that these markers will be valuable for the diagnosis of metastases from several if not many types of cancer.
  • the present invention relates to the use of a protein expression pattern (or profile or "signature") which is clinically relevant to metastasizing lung cancer.
  • a protein expression pattern or profile or "signature” which is clinically relevant to metastasizing lung cancer.
  • the identity of proteins that are correlated with lung cancer metastasis are provided.
  • the expression profile whether embodied in nucleic acid expression, protein expression, or other expression formats, may be used to predict survival of subjects afflicted with lung cancer and the likelihood of cancer recurrence.
  • the invention thus provides for the use of a gene expression pattern (or profile or
  • the invention thus provides for the use of the gene expression pattern which correlates with the recurrence of cancer at the same location and/or in the form of metastases.
  • the pattern is able to distinguish patients with cancer into at least those with good or poor survival outcomes.
  • the present invention provides a non-subjective means for the identification of patients with cancer, in particular lung cancer, as likely to have a good or poor survival outcome by assaying for the expression pattern disclosed herein.
  • the present invention provides an objective expression pattern, which may be used alone or in combination with subjective criteria to provide a more accurate assessment of cancer patient outcomes, including survival and the recurrence of cancer.
  • the expression pattern of the invention thus provides a means to determine lung cancer prognosis as well as cancer prognosis in general.
  • the expression pattern comprises the proteins identified in supplementary tables 1-3, which pattern is capable of discriminating between lung cancer outcomes with significant accuracy.
  • the genes are identified as correlated with various lung cancer outcomes such that the levels of their expression are relevant to a determination of the preferred treatment protocols, of a lung cancer patient.
  • the invention provides a method to determine the outcome of a subject afflicted with, or suspected of having, lung cancer by assaying a cell containing sample from said subject for expression the proteins identified in supplementary tables 1-3 as correlated with lung cancer outcomes.
  • a profile of proteins that are highly correlated with one outcome relative to another may be used to assay a sample from a subject afflicted with, or suspected of having, lung cancer to predict the outcome of the subject from whom the sample was obtained. Such an assay may be used as part of a method to determine the therapeutic treatment for said subject based upon the lung cancer outcome identified.
  • the correlated proteins identified in supplementary tables 1-3 are used in combination to increase the ability to accurately correlating a molecular expression phenotype with a lung cancer outcome. This correlation is a way to molecularly provide for the determination of survival outcomes as disclosed herein.
  • an assay may utilize any identifying feature of an identified individual gene as disclosed herein as long as the assay reflects, quantitatively or qualitatively, expression of the gene in the "transcriptome” (the transcribed fraction of genes in a genome) or the
  • proteome (the translated fraction of expressed genes in a genome). Identifying features include, but are not limited to, unique nucleic acid sequences used to encode (DNA), or express (RNA), said gene or epitopes specific to, or activities of, a protein encoded by said gene.
  • the present invention is directed to a method for diagnosing/determining the risk of a (lung) cancer patient for developing metastases by establishing a differential expression of the proteins identified in supplementary tables 1-3.
  • the present invention provides for an array for prediction of risk of metastasis in lung cancer having probes specific for the proteins identified in supplementary tables 1-3.
  • a protein expression "pattern” or “profile” or “signature” refers to the relative expression of the proteins identified in supplementary tables 1-3 between two or more lung cancer survival outcomes which is correlated with being able to distinguish between said outcomes.
  • a "gene” is a polynucleotide that encodes a discrete product, whether RNA or proteinaceous in nature. It is appreciated that more than one polynucleotide may be capable of encoding a discrete product.
  • the term includes alleles and polymorphisms of a gene that encodes the same product, or a functionally associated (including gain, loss, or modulation of function) analog thereof, based upon chromosomal location and ability to recombine during normal mitosis.
  • correlate or “correlation” or equivalents thereof refer to an association between expression of genes and a physiologic state of a lung cell to the exclusion of one or more other state as identified by use of the methods as described herein.
  • a gene may be expressed at higher or lower levels and still be correlated with one or more lung cancer state or outcome.
  • a "polynucleotide” is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications including labels known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as unmodified forms of the polynucleotide.
  • uncharged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • amplify is used in the broad sense to mean creating an amplification product can be made enzymatically with DNA or RNA polymerases.
  • Amplification generally refers to the process of producing multiple copies of a desired sequence, particularly those of a sample.
  • protein is used to describe a chain of amino acids as encoded for by the gene or polynucleotide. It also encompasses all naturally occurring modification products (e.g. proteins which have had a leader sequence removed, post-translational modification products and degradation products).
  • differentiated protein refers to a protein whose expression is activated to a higher or lower level in a subject suffering from cancer, such as lung cancer, relative to its expression in a normal or control subject.
  • the terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease.
  • differentiated protein expression is considered to be present when there is at least an about two-fold, preferably at least about three-fold, more preferably at least about four-fold, most preferably at least about six-fold difference between the expression of a given protein in normal and diseased subjects, or in various stages of disease development in a diseased subject.
  • either particular proteins may be used for reference that are not affected by the same regulatory sequences, whose level of expression remains constant in the particular disease, state or disorder to be examined, so that the amount of expression can serve as a background level to be used for comparative purposes, to determine if a particular gene is turned on or off in that disease, disorder or other state to be examined.
  • the expression of individual proteins may be compared to the expression levels of total protein.
  • the microarray also include "housekeeping" proteins (control proteins), o r proteins that are not affected by the same regulatory sequences, whose level of expression remains constant in the particular disease, state or disorder to be examined, so that the amount of expression can serve as a background level to be used for comparative purposes, to determine if a particular gene is turned on or off in that disease, disorder or other state to be examined.
  • control proteins control proteins
  • o r proteins that are not affected by the same regulatory sequences, whose level of expression remains constant in the particular disease, state or disorder to be examined, so that the amount of expression can serve as a background level to be used for comparative purposes, to determine if a particular gene is turned on or off in that disease, disorder or other state to be examined.
  • a "microarray” is a linear or two-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support such as, but not limited to, glass, plastic, or synthetic membrane. The density of the discrete regions on a microarray is determined by the total numbers of immobilized proteins to be detected on the surface of a single solid phase support.
  • a protein microarray is an array of antibodies placed on a chip or other surfaces used to bind proteins from a sample.
  • label refers to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule, such as a differentially expressed protein of the present invention. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such , a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • support refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports, such as glass slides.
  • lung tissue sample or “lung cell sample” refers to a sample of lung tissue or fluid isolated from an individual suspected of being afflicted with, or at risk of developing, lung cancer.
  • Detection includes any means of detecting, including direct and indirect detection of protein expression and changes therein. For example, “detectably less” products may be observed directly or indirectly, and the term indicates any reduction (including the absence of detectable signal). Similarly, “detectably more” product means any increase, whether observed directly or indirectly.
  • prognosis is used herein to refer to the prediction of the likelihood of cancer- attributable death or progression, including recurrence and metastatic spread.
  • prediction is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following surgical removal or the primary tumor and/or chemotherapy for a certain period of time without cancer recurrence.
  • the predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
  • the predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities is likely.
  • the present invention relates to the identification and use of a protein expression pattern (or profile or "signature") based on the proteins identified in supplementary tables 1-3 which discriminates between (or are correlated with) lung cancer survival and recurrence outcomes in a subject.
  • a protein expression pattern or profile or "signature”
  • Expression based on detection of a presence, increase, or decrease in protein levels or activity may be performed by any electrophoretic based, immunohistochemistry (IHC) based, blood based (especially for secreted proteins), antibody (including autoantibodies against the protein) based, exfoliate cell (from the cancer) based, mass spectroscopy based, and image (including used of labeled ligand) based method known in the art and recognized as appropriate for the detection of the protein.
  • IHC immunohistochemistry
  • blood based especially for secreted proteins
  • antibody including autoantibodies against the protein
  • exfoliate cell from the cancer
  • mass spectroscopy based mass spectroscopy based
  • image including used of labeled ligand
  • a preferred embodiment using a nucleic acid based assay to determine expression is by immobilization of one or more sequences of the genes encoding the proteins identified herein on a solid support, including, but not limited to, a solid substrate as an array or to beads or bead based technology as known in the art.
  • a solid support including, but not limited to, a solid substrate as an array or to beads or bead based technology as known in the art.
  • solution based expression assays known in the art may also be used.
  • the immobilized genes may be in the form of polynucleotides that are unique or otherwise specific to the genes such that the polynucleotides would be capable of hybridizing to a DNA or RNA corresponding to the genes.
  • the present invention provides a more objective set of criteria, in the form of gene expression profiles of a discrete set of genes, to discriminate (or delineate) between lung cancer outcomes.
  • the assays are used to discriminate between good and poor outcomes within 5, or about 5, years after surgical intervention to remove lung cancer tumors.
  • a "good” outcome may be viewed as a better than 50% survival rate after about 60 months post surgical intervention to remove lung cancer tumor(s).
  • a “good” outcome may also be a better than about 60%, about 70%, about 80% or about 90% survival rate after about 60 months post surgical intervention.
  • a “poor” outcome may be viewed as a 50% or less survival rate after about 60 months post surgical intervention to remove lung cancer tumor(s).
  • a “poor” outcome may also be about a 70% or less survival rate after about 40 months, or about a 80% or less survival rate after about 20 months, post surgical intervention.
  • the isolation and analysis of a lung cancer cell sample may be performed as follows:
  • Procedure is performed on a patient to obtain a sample.
  • those cells are harvested (e.g. by microdissection).
  • Proteins are purified and optionally labeled.
  • the proteins are contacted with a microarray containing antibodies against the proteins identified herein as correlated to discriminations between lung cancer outcomes under suitable antibody-recognition conditions.
  • the microarray may optionally be treated with a second antibody for indirect immunodetection to increase sensitivity. (9) Then the array is processed and scanned to obtain a pattern of intensities of each spot (relative to a control for general protein expression in cells) which determine the level of expression of the proteins in the cells.
  • the pattern of intensities is analyzed by comparison to the expression patterns of the proteins in known samples of lung cancer cells correlated with outcomes (relative to the same control).
  • the prognosis may be derived with reference to a combination of individual proteins detected in the array in order to increase the sensitivity or specificity of the assay.
  • Krt ⁇ keratin 8; Krt14, keratin 14 Krt18, keratin 18; Krt19, keratin 19; Ywhae, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide; Ywhah, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; Ywhaz, tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide; Psma5, proteasome (prosome, macropain) subunit, alpha type 5; Psma ⁇ , proteasome (prosome, macropain) subunit, alpha type 6; Psmb2, proteasome (prosome, macropain) subunit, beta type 2; Psm
  • CMT167, CMT64 and CMT170 with highest, middle or lowest metastatic potential were used and designated as CMT167(H), CMT64(M) and CMT170(L) hereafter.
  • Cells were cultured in DMEM with 4.5g/L glucose (Gibco Invitrogen) supplemented with 10% FBS (Gibco Invitrogen), 1 % (v/v) L-Glutamax I (Gibco Invitrogen) and 0.5% penicillin and streptomycin (Gibco Invitrogen) in a humidified incubator at 37 0 C and 5% CO 2 .
  • H BSS Hank's buffered salt solution
  • %IOD and standard deviation were calculated. F-test was performed to test whether the variance of protein spot %IOD from triplicate samples in two groups is equal or unequal, and P > 0.01 was regarded as equal variance. Then changes in the expression between two comparable groups were analyzed by Student's t-test and p ⁇ 0.01 was regarded as significant.
  • Protein spots of interest were excised manually from the preparative gel, alkylated with iodoacetamide, in-gel digested with trypsin as previously described [Proteomics 2007, 7, 2340-2349] and analyzed by MALDI-TOF/TOF MS on either a 4700 Proteomics Analyser
  • MS and MS/MS data were processed by the appropriate software including FlexAnalysis 2.2(Bruker), BioTools 2.2(Bruker), m/z (ProteoMetrics) and Data explorer (Applied Biosystems, version 4.4). MS and MS/MS data from each individual spot were combined and used for database searching. Protein identification was performed using MASCOT software (http://www.matrixscience.com version 2.1 ) to search for mouse species sequences in the NCBInr database. The following parameters were used for database searches: fixed modification: carbamidomethylation of cysteine; variable modification: oxidation of methionine; monoisotopic mass accuracy ⁇ 70 or IOOppm for the 4700 and
  • Bioinformatics analysis of identified proteins was performed by the DAVID software, i.e. the d a t a b a s e f o r a n n o t a t i o n , v i s u a l i z a t i o n a n d i n t e g r a t e d i s c o v e r y (http://david.abcc.ncifcrf.gov/) [Genome Biol. 2003, 4, 3].
  • CMT cell lines at passage 5, 15, 35 were labelled with [ 35 S]-methionine in triplicate using the previously described optimized conditions.
  • Three independent gels were made from each cell line at each passage and analyzed to allow statistical analysis of the data.
  • 2-DE gel phosphoimages were analyzed by the ProteoMiner image analysis program resulting in reproducible observation of 2,397 protein spots in each gel.
  • a representative gel image is shown in Fig. 1. Pair wise comparison was made between the CMT cell lines (CMT167(H) Vs. CMT170(L); CMT64(M) Vs. CMT170(L); CMT167(H) Vs. CMT64(M)) at passage 5, 15 and 35 respectively.
  • CMT167(H) and CMT170(L) at one or both of the passages (Fig. 3). Many of them showed a consistent trend in either down- or up-regulation through all three cell lines with CMT64 in most cases having an expression level between CMT167(H) and CMT170(L) (categories A and B in Fig. 3: down- or up-regulated respectively). The tendency was most clear at passage 5 where the expression level of protein from CMT167(H) was always significantly higher than CMT170(L) (category A) or significantly lower (category B). For a number of protein spots the trend in change pattern did not seem to reflect the metastatic potential from high over medium to low (category C in Fig. 3). The identified proteins, their change pattern at passage 5 and their reported functions related to cancer and metastasis are given in Table 2.
  • the identified proteins which were differentially expressed between the cell lines at passage 5 represent 82 unique proteins.
  • Krt34 and Lmna were classified to be involved in cell communication pathway, five (Psma ⁇ , Psma ⁇ , Psmb2, Psmc2and Psmd14) were classified to the proteasome pathway and six (Mcm7, Pcna, Skpi a, Ywhae, Ywhaz and Ywhah) to the cell cycle pathway.
  • Mcm7, Pcna, Skpi a, Ywhae, Ywhaz and Ywhah to the cell cycle pathway.
  • a total of 40 unique proteins were differentially expressed between three cell lines. Of these, four proteins (Krt7, Krt14, Krt19 and Vim) were found to belong to the cell communication pathway.
  • Psmc2 and Ywhah involved in the proteasome and the cell cycle pathways respectively.
  • Figure 1 shows a representative 2-DE of CMT167(H). The encircled spots were differentially expressed between one or more of the cell lines at passage 5.
  • Figure 2 shows: (A) Section of the 2-DE image showing spots 699 and 1890 which were down-regulated and spot 736 which was up-regulated in CMT167(H) compared to CMT170(L); (B) The MALDI-TOF spectra obtained from spot 736; (C) MALDI TOF/TOF spectra obtained from the precursor ion at m/z 1362.79 in the spectrum shown in (B).
  • the protein could be identified as keratin 7 based on the peptide mass fingerprint in B and confirmed by an almost the full amino acid sequence of the peptide 182 TAAENEFVLLKK 193 .
  • FIG. 3 shows expression level of protein spots identified in CMT167(H) and CMT170(L) at passage 5 in all of three CMT cell lines at both passage 5 and 15. Results are shown as
  • IOD% mean ⁇ S.D. of three independent gels.
  • A Protein spots down-regulated from the highest metastatic cell line CMT167(H) over moderate metastatic CMT64(M) to the lowest metastatic CMT170(L).
  • B Protein spots up-regulated from the highest metastatic CMT167(H) over moderate metastatic CMT64(M) to the lowest metastatic CMT170(L).
  • C Protein spots without a consistent change trend in three CMT cell lines.
  • Lung cancer is one of the leading causes of cancer-related deaths in the world. It is grouped in small-cell lung cancer and non-small-cell lung cancer. The latter represents about 80% of human lung cancers and can be subdivided into adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma [The lung in Pathologic basis of diseases, Elsevier Inc,
  • the genetic changes during lung cancer development includes the activation of oncogenes such as myc, Kras, EGFR etc and inactivation of tumor suppressor genes including P53, P16 and Rb etc [Biochim. Biophys. Acta. 1998, 1378, F21-
  • mouse models have been widely used for studying carcinogenesis of human lung cancers and many of the major genetic alterations detected in human lung cancers have also been identified in mouse lung tumors
  • Metastasis is the most lethal attribute of cancer and presents a continuing diagnostic and therapeutic challenge.
  • the molecular mechanisms behind metastasis are highly complex and poorly understood, and elucidation of these is highly desirable.
  • the advent of three almost identical isogenic cell lines with demonstrated differences in metastatic potential might throw new light on the metastasis phenomenon.
  • CMT170(L) cell lines have been compared in terms of genetic composition and mRNA levels [Cancer Res. 2000, 60, 1 1 73-1176].
  • three mouse lung adenocarcinoma CMT cell lines with different metastatic potential are used to screen for proteins which might be involved in metastasis. Such proteins might also in the future be used as candidates for further discovery of metastatic markers.
  • the intermediate filament (IF) cytoskeleton of all epithelia is built from heteropolymers of type I and type Il keratins with 1 :1 molar ratio.
  • Various keratin combinations are expressed in an epithelial cell-type preferential manner and their abnormal expression has been linked to human diseases [Lung cancer 2007, 55, 295-302].
  • Keratin typing has become a major tool in tumor and histodiagnosis providing molecular parameters to assess the differentiation status of various types of carcinomas [Exp. Cell Res. 2007, 313, 2021-2032].
  • KRT7, KRT8, KRT18 and KRT19 are the main cytokeratins.
  • six cytokeratins including Krt7, Krt8, Krt14, Krt18, Krt19 as well as one type I hair keratin Krt34 which are differentially expressed between CMT cell lines.
  • Krt18 identified from spot 871 and 870 showed more than 50% down- regulation in higher metastatic CMT cell lines (CMT167(H) and CMT64(M)) compared to the low metastatic cell line CMT170(L) (Table 2, Fig. 3 and Suppl. Table 1 .).
  • Decreased expression of KRT18 was also observed in a breast cancer cell line with higher metastatic potency [Clin. Cancer Res. 1996, 2, 1879-1885] and overexpression of KRT18 in breast cancer cells inhibited metastasis both in vitro and in vivo [MoI. Cancer Res. 2005, 3, 365-
  • Krt8 is the partner of Krt18. Its elevated expression had been related to increased metastasis in NSCLC [Lung cancer 2002, 38, 31-38] and melanoma [Cancer Metastasis Rev. 1996, 15, 507-525], however, reduction of KRT8 was also linked to increased aggressiveness in colorectal cancers [Ce//. Oncol. 2006, 28, 167-175]. In our study, the expression pattern of Krt8 is somewhat complex. Krt8 identified from spot 786 was 2.3 fold higher in CMT167(H) than in CMT170(L) (Table 2 and Fig.
  • Krt8 identified from spot 3398 and 3399 indicated that Krt8 was down regulated in CMT64(M) compared to CMT170(L) (Suppl. Table 1 ).
  • the position of these three spots on 2-DE image is very close and their molecular weight is close to 54 kDa in accordance with full length Krt8.
  • Previously an aberrantly spliced or degraded 45 kDa Krt8 have been reported and found in NSCLC and aggressive breast cancer [Lung Cancer 2003, 42, 153-161 ; Cancer Sci. 2003, 94, 864- 870]. This isoform of Krt8 was not observed in the present study, most likely because its expression level did not fulfil our criteria for spot selection.
  • Krt14 and Krt19 are a useful diagnotic marker for metastasis in different tumors such as NSCLC, head and neck carcinoma and hepatocellular carcinoma
  • 14-3-3 family of proteins in lung cancer metastasis 14-3-3 Proteins are a family of highly conserved 30 kDa acidic proteins involved in the cell cycle pathway. They are expressed in all eukaryotic cells and seven isoforms including YWHAB, YWHAG, YWHAE, YWHAZ, YWHAH, SFN and YWHAQ have been identified in mammals. Accumulating evidence indicates that the 14-3-3 proteins play an important role in the regulation of cell proliferation, differentiation and transformation via binding proteins involved in signal transduction, cell cycle control, vesicular transport, DNA replication and apoptosis [Annu. Rev. Pharmacol. Toxicol. 2000, 40, 617-647; Bioessays 2001 , 23, 936- 946].
  • 26S proteasome In eukaryotic cells, the 26S proteasome is a 2.5MDa multisubunit protein complex and is the site for ATP dependent degradation of ubiquitin-tagged proteins in the cytosol and nucleus. It is composed of two major subunits including the 2OS catalytic core complex and the 19S regulatory particle [Annu. Rev. Biochem. 1999, 68, 1015-1068].
  • the controlled degradation is crucial in maintaining a variety of cellular processes such as transcription, DNA repair, cell cycle and apoptosis. Disruption of this pathway has been implicated in several diseases. In cancers, abnormal proteasomal degradation could either enhance the effect of oncoproteins or reduce the level of tumor suppressor proteins.
  • Group 1 , 2 and 3 correspond to comparison groups CMT167(H) Vs. CMT170(L), CMT64(M) Vs. CMT170(L) and CMT167(H) Vs. CMT64(M) respectively.
  • Numbers listed here are number of changed spots (P ⁇ 0.01 , 50% up- or down-regulation), the number of successful identifications and the number of unique proteins identified respectively.
  • Vps24 vacuolar protein sorting 24 (yeast) 0.082 0.010 0.047 0.001 1.75 8 24% 2 168 NP_080059
  • Vps29 vacuolar protein sorting 29 (S. pombe) 0.013 0.002 0.004 0.001 3.01 4 20% 1 154 NP_062754
  • MS information including number of matched peptides, sequence coverage, number of peptide ions sequenced by MS/MS and mascot score are listed in A, B, C, D respectively.
  • a 786 keratin 8 2.35 1.74 1.35 NP_112447 Mam cytokeratm in non-small cell lung cancer [20].
  • RNA exonuclease 2 homolog 1.62 1.36 1.19 NP_077195 NR (S.cerevisiae)
  • proteasome prosome, macropain subunit
  • alpha 3.11 1.18 2.64 NP_036098 Upregulated in hepatocellular carcinomas of p21 -HBx type 6 transgenic mice [24].
  • NP_031663 Upregulation may be related to lymph node metastasis in esophageal squamous cell carcinoma [38].
  • NR represents proteins not reported to be directly related to cancer or metastasis according to our literature search. [ND is usually used for Not
  • Vps24 vascuolar protein sorting 24 (yeast) 0.068 0.002 0.038 0.000 1.78 8 24% 2 168 NP_080059
  • Gdi2 guanosine diphosphate (GDP) 0.094 0.003 0.055 0.006 1.71 18 44% 1 201 NP_032138 dissociation inhibitor 2

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Abstract

L'invention concerne un profil d'expression de protéine ou de gène avec une pertinence clinique à une métastase cancéreuse. En particulier, l'invention concerne des identités de protéines qui sont corrélées à une métastase de cancer de poumon. Les profils d'expression de protéine peuvent être utilisés pour prédire la survie de sujets atteints d'un cancer et pour prédire une récurrence du cancer. Les profils peuvent être également utilisés dans l'étude et/ou le diagnostic de cellules cancéreuses et de tissus cancéreux, comprenant la graduation d'un cancer du poumon invasif, ainsi que pour l'étude et/ou la détermination d'un pronostic d'un patient. Lorsqu'ils sont utilisés pour un diagnostic ou un pronostic, les profils sont utilisés pour déterminer le traitement du cancer sur la base de la probabilité d'espérance de vie et de métastase.
PCT/EP2009/053923 2008-04-03 2009-04-02 Marqueurs métastatiques WO2009121924A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2397564A1 (fr) * 2010-06-15 2011-12-21 Sysmex Corporation Procédé pour déterminer la métastase de ganglions lymphatiques dans un cancer du poumon, appareil pour déterminer la métastase de ganglions lymphatiques dans un cancer du poumon et produit de programme informatique
WO2014061419A1 (fr) * 2012-10-17 2014-04-24 公立大学法人奈良県立医科大学 Marqueur de cancer innovant et son utilisation
CN110873799A (zh) * 2019-12-09 2020-03-10 四川大学华西医院 Psmb4自身抗体检测试剂在制备肺癌筛查试剂盒中的用途
CN113552357A (zh) * 2021-07-23 2021-10-26 燕山大学 白三烯a4水解酶作为肺癌早期标志物的应用

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ZHANG K ET AL: "Comparative proteome analysis of three mouse lung adenocarcinoma CMT cell lines with different metastatic potential by two-dimensional gel electrophoresis and mass spectrometry", PROTEOMICS 200812 DE, vol. 8, no. 23-24, December 2008 (2008-12-01), pages 4932 - 4945, XP002530748, ISSN: 1615-9853 1615-9861 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2397564A1 (fr) * 2010-06-15 2011-12-21 Sysmex Corporation Procédé pour déterminer la métastase de ganglions lymphatiques dans un cancer du poumon, appareil pour déterminer la métastase de ganglions lymphatiques dans un cancer du poumon et produit de programme informatique
WO2014061419A1 (fr) * 2012-10-17 2014-04-24 公立大学法人奈良県立医科大学 Marqueur de cancer innovant et son utilisation
JPWO2014061419A1 (ja) * 2012-10-17 2016-09-05 公立大学法人奈良県立医科大学 新規癌マーカーおよびその利用
US9857375B2 (en) 2012-10-17 2018-01-02 Public University Corporation Nara Medical University Cancer marker and utilization thereof
CN110873799A (zh) * 2019-12-09 2020-03-10 四川大学华西医院 Psmb4自身抗体检测试剂在制备肺癌筛查试剂盒中的用途
CN113552357A (zh) * 2021-07-23 2021-10-26 燕山大学 白三烯a4水解酶作为肺癌早期标志物的应用

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