WO2009082744A2 - Pronostic et traitement véhiculé par interférence du cancer du sein - Google Patents

Pronostic et traitement véhiculé par interférence du cancer du sein Download PDF

Info

Publication number
WO2009082744A2
WO2009082744A2 PCT/US2008/088065 US2008088065W WO2009082744A2 WO 2009082744 A2 WO2009082744 A2 WO 2009082744A2 US 2008088065 W US2008088065 W US 2008088065W WO 2009082744 A2 WO2009082744 A2 WO 2009082744A2
Authority
WO
WIPO (PCT)
Prior art keywords
mir
cells
breast cancer
expression
oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2008/088065
Other languages
English (en)
Other versions
WO2009082744A3 (fr
Inventor
Joan Massague
Sohail F. Tavazoie
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Memorial Sloan Kettering Cancer Center
Original Assignee
Memorial Sloan Kettering Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Memorial Sloan Kettering Cancer Center filed Critical Memorial Sloan Kettering Cancer Center
Publication of WO2009082744A2 publication Critical patent/WO2009082744A2/fr
Publication of WO2009082744A3 publication Critical patent/WO2009082744A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57515—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the breast
    • 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

  • metastasis is the overwhelming cause of mortality in patients with solid tumours, the understanding of its molecular and cellular determinants is limited. Fidler, I. J. The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nature Rev. Cancer 3, 453 458 (2003); Weigelt, B., Peterse, J. L. & van't Veer, L. J. Breast cancer metastasis: markers and models. Nature Rev. Cancer 5, 591 602 (2005); Gupta, G. P. & Massague, J. Cancer metastasis: building a framework. Cell 127, 679 695 (2006).
  • Transcriptional profiling has revealed sets of genes, or 'signatures', for which expression in primary tumours correlates with metastatic relapse or poor survival.
  • MicroRNAs are attractive candidates as upstream regulators of metastatic progression because miRNAs can post-transcriptionally regulate entire sets of genes.
  • a genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumours.
  • MicroRNA expression profiles classify human cancers. Nature 435, 834-838 (2005), and interfering with miRNA processing enhances experimental tumorigenesis. Kumar, Nature Genet. These findings suggest that this class of regulators contains suppressors of tumour progression and possibly metastasis.
  • the invention provides a method for treating metastatic breast cancer comprising administering to a patient diagnosed with breast cancer an agent, said agent
  • oligonucleotide that reduces the expression of a gene in cancerous breast cells exposed to the oligonucleotide wherein the gene is overexpressed in both bone and lung metastases of breast cancer, and is down-regulated by miR-335, miR-126 and/or miR-206 and wherein the oligonucleotide has a sequence that is complementary to a portion of said gene or a transcript of said gene;
  • (b) comprising an oligonucleotide that increases the amount of miR-335, miR-126 and/or miR-206, thereby treating the metastatic breast cancer.
  • RNA or protein that are the product of one or more genes selected from the group consisting of SRY-box containing transcription factor 4 (SOX4), Tenascin C (TNC), receptor-type tyrosine protein phosphatase (PTPRN), collagen, type I, alpha 1 (COLl Al), Mer receptor tyrosine kinase (MERTK), and phospholipase C beta 1 (PLCBl); comparing said diagnostic signature to a control signature; and based on the comparison, giving a prognosis of high risk for metastasis if the difference between the diagnostic signature and the control signature is greater than a threshold.
  • Observed levels for miRNA selected from the from among miR-335, miR-126, and/or miR-206 can also be used for diagnostic purposes.
  • the invention also provides for the use of the diagnostic method in conjunction with the treatment method. This permits more aggressive treatment where a breast cancer is one that has a greater risk of metastases.
  • an oligonucleotide in the manufacture of a medicament for the treatment of metastatic breast cancer; wherein said oligonucleotide has a sequence that reduces the expression of a gene in cancerous breast cells exposed to the oligonucleotide; wherein the gene is overexpressed in both bone and lung metastases of a source cell; and wherein the oligonucleotide has a sequence that is complementary to a portion of said gene or a transcript of said gene.
  • composition comprising a pharmaceutically acceptable carrier, and an oligonucleotide that reduces the expression of human Tenascin C (Seq. ID No. 10).
  • the agent is an oligonucleotide comprising a sequence selected from the group consisting of Seq. ID No. 4, Seq. ID No. 5, Seq. ID No. 6, and Seq. ID No. 7.
  • kits for detection and characterization of breast cancer comprising reagents in packaged combination specifically for detecting or quantifying two or more markers selected from the group consisting of SRY-box containing transcription factor 4 (SOX4), Tenascin C (TNC), receptor-type tyrosine protein phosphatase (PTPRN), collagen, type I, alpha 1 (COLl Al), Mer receptor tyrosine kinase (MERTK), phospholipase C beta 1 (PLCBl), miR-335, miR-126 and miR-206.
  • SOX4 SRY-box containing transcription factor 4
  • TTC Tenascin C
  • PPRN receptor-type tyrosine protein phosphatase
  • collagen type I
  • alpha 1 alpha 1
  • MERTK Mer receptor tyrosine kinase
  • PLCBl phospholipase C beta 1
  • Figs. IA-D show systematic identification of miRNAs that suppress lung and bone metastasis in multiple human breast cancer cell derivatives.
  • Figs. 2A-C show miR-126 suppresses overall tumour growth and proliferation whereas miR-335 and miR-206 regulate migration and morphology.
  • Figs. 3A-B show clinical association of miR-335 and miR-126 with metastasis-free survival.
  • Figs. 4A-E show a miR-335-regulated gene set includes SOX4 as a miR-335 direct target.
  • Figs. 5A-C show miR-335 regulates metastasis and invasion through suppression of SOX4 and TNC.
  • Fig. 6 shows stem-loop quantitative RT-PCR for the mature forms of the downregulated miRNAs identified through hybridization profiling.
  • Fig. 7 shows quantification of miRNA expression in LM2 cells with restored expression of miRNAs.
  • Fig. 8 shows bioluminescence quantification of lung colonization by additional miRNAs that are downregulated in metastatic cells.
  • Figs. 9A-C show loss of metastasis suppressor miRNA expression from primary human cancer cells as well as exogenously introduced vector subsequent to metastatic progression.
  • Fig. 10 shows miR-126 suppression of primary tumour growth.
  • Figs. HA-B show immunohistochemical analysis for apoptotic and proliferation markers in mammary tumours.
  • Fig. 12 shows restoration of miR-335 and miR-206 expression in metastatic cells reduces the fraction of elongated cells.
  • Fig. 13 shows immunohistochemical analysis of the mesenchymal marker vimentin in LM2 cells expressing miR-335, miR-126, or miR-206.
  • Fig. 14 shows quantification of miRNA levels in human breast tumour samples. Each vertical bar represents expression levels of metastasis suppressor miRNAs miR-335, miR- 206, and miR-126 in addition to miR-122a and miR-199a.
  • Fig. 15 shows the association between the loss of miR-206 expression in primary breast tumours and metastatic relapse.
  • Figs. 16A-B show the miR-335 regulated genes SOX4 and TNC alter the morphology of lung metastatic cells and suppress migration.
  • Figs. 17A-B show the miR-335 regulated six-gene signature predicts overall metastasis-free survival in the MSK-82 and EMC-286 datasets.
  • Fig. 18 shows miR-335 RNA mimetic delivery to breast cancer cells specifically suppresses the expression of metastasis promoter gene SOX4 similarly as virally expressed miR-335.
  • Fig. 19 shows miR-126 RNA mimetic delivery to breast cancer cells suppresses proliferation of metastatic cells similarly as virally expressed miR-126.
  • RNA or protein the products of the six genes, and/or levels of one or more of the three miRNA provides a prognostic/diagnostic indicator of the likelihood or presence of breast cancer metastases.
  • agents that decrease the amount of protein gene product and/or decrease the amount of the relevant miRNA can be used in the treatment of metastatic breast cancer.
  • oligonucleotides means a short sequence of nucleic acids, no more than 200 bases long.
  • An oligonucleotide may be made of DNA, RNA or a combination, and may be chemically modified to increase stability.
  • complementary refers to the base pairing relationshiop between strands of nucleic acids arranged in an antiparallel manner, such that the strands will hybridize with sufficient strength under intracellular conditions to change expression of a gene.
  • the terms 'treatment' or 'treating' refer to the application of an agent with the intent to produce a benefit to a patient being treated.
  • a benefit need not be a complete or permanent cure, but may be only a lessening of the rate at which tumorigenesis is occurring, thereby delaying onset or progression of metastases, particularly to bone and lung.
  • such treatment need not in actuality produce the beneficial, as long is it is given with the intention of obtaining a result.
  • gene product refers to the product of transcription or translation of a gene sequence. This may include RNA or protein products, including or regardless of later processing or conformational changes.
  • 3'-UTR or "3'-untranslated region” is a particular section of messenger RNA (mRNA) that follows the coding region. This region contains transcription and translation regulating sequences.
  • mRNA messenger RNA
  • genetic signature refers to the numerical value of the level of expression of one or more genes in a tissue sample.
  • a genetic signature can be based on a single gene/gene product, or it may be a composite of values for multiple genes/gene products.
  • control signature means an average genetic signature obtained from measurements of multiple samples from non-metastasizing breast cells (normal or cancerous). The sample size should be large enough to be statistically relevant. It will be apparent to one skilled in the art that a control signature and the genetic signature to which it is compared are obtained using the same test conditions, although they need not be done at the same time.
  • RNA short hairpin RNA. It is a sequence of
  • RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA hairpin structure is cleaved by the cellular machinery and are precursors to miRNA.
  • miRNAs are single-stranded RNA molecules generally of less than 24 nucleotides in length excised from 60- to 110-nucleotide shRNA precursors involved in the regulation of expression of protein-coding genes (PCGs), which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre- miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. miR-335 has the sequence set forth in Seq. ID No. 11. miR-126 has the sequence set forth in Seq. ID No. 12. miR-206 has the sequence set forth in Seq. ID No. 13.
  • the genes were SRY-box containing transcription factor 4 (SOX4), Tenascin C (TNC), receptor-type tyrosine protein phosphatase (PTPRN), collagen, type I, alpha 1 (COLl Al), Mer receptor tyrosine kinase (MERTK), and holipase C beta 1 (PLCBl).
  • SOX4 SRY-box containing transcription factor 4
  • TMC Tenascin C
  • PPRN receptor-type tyrosine protein phosphatase
  • collagen type I
  • alpha 1 COLl Al
  • MERTK Mer receptor tyrosine kinase
  • PLCBl holipase C beta 1
  • One embodiment of the present invention is a method for diagnosing metastatic potential of breast cancer for a patient, through the determination of a genetic signature and comparison of that signature to a control signature, hi accordance with the invention, this can be accomplished through a determination of the amount of one or more markers, wherein the markers are gene products (protein or RNA) selected from the group consisting of SOX4, TNC, PTPRN, COLlAl), MERTK and PLCBl, and/or one or more micro RNAs selected from the gorup consisting of miR-335, miR-126, and miR-206.
  • the marker(s) include SOX4.
  • the marker(s) include TNC.
  • al six proteins are used as markers
  • hi specific embodiments, miR- 335 and/or miR-126 are used as markers.
  • the genetic signature is obtained on a sample of breast cancer cells obtained from a patient. These can be metastatic cells or cells that have not yet undergone metastases, hi either case, a characterization of the metastatic potential of the cells is obtained which can be used in counseling and selection of treatment options, including the selection or very aggressive treatment or no treatment where the cancer has a high metastatic potential.
  • the genetic signature is compared to a control signature obtained from non-cancerous breast cells.
  • a protein marker is present at higher than a threshold level, or an miRNA marker is present at lower than a threshold level, then the breast cancer from the patient is considered to be one with high metastatic potential.
  • the determination of specific numerical values for the threshold is dependent on the particular tests that are included in the formation of the signatures, and the level of risk that is to be assigned as high risk.
  • the threshold level is suitably the sum of the six expression levels of the control signature plus one standard deviation for the control signature.
  • Detection of the gene products or miRNAs may be done using any of various known methods from detection or proteins or RNA, including without limitation: a) polymerase chain reaction (PCR) technology, for example quantitative PCR of one or more nucleic acids, preceded by retro-transcription in case the sample to be analyzed is RNA, another example is stem-loop quantitative RT-PCR which is a modification of RT-PCR specially adapted to the amplification and quantification of micro RNAs, b) nucleic acid hybridization technologies, for example nucleic acid gel electrophoresis followed by transfer to a membrane and posterior hybridization (Northern/Southern blot), or hybridization to membranes where the nucleic acid is deposited (purified or not or as a fraction) directly to a membrane (slot or dot blot), or direct hybridization into tissue or cells mounted on a slide; c) microarray technology.
  • PCR polymerase chain reaction
  • a profile can also be obtained by determining the presence, absence or quantity of a specific protein or proteins, and such profiling can be done using methods well known in the art like ELISA, immunohistochemistry or Western blot, that use polyclonal or monoclonal antibodies able to recognize specific proteins.
  • breast cancer cells for analysis according to the methods of the present invention.
  • One such method is biopsy, removing a sample of breast tissue containing cancer. Further the tissue can be separated into cancer cells and non-cancer cells and the cancer cells processed for further analysis of DNA, RNA or protein.
  • Another method is obtaining cancer cells from bodily fluids for example ductal lavages or blood according to methods known in the art.
  • An embodiment of the present invention provides a method to provide to a breast cancer patient a prognosis of metastasis-free survival consisting of obtaining breast tumor cells from that breast cancer patient, determining the RNA or protein levels of at least one of the following: COLlA gene, PTPRN gene, MERKT gene, PLCBl gene, TNC gene and SOX4 gene, determining an aggregate expression score based on the expression of at least those genes or proteins, the miR-335-signature, and considering those patients whose expression scores exceed one standard deviation from the mean to be miR-335-signature- positive and therefore to have worse metastasis-free survival prognosis than those patients that are not miR-335- signature-positive.
  • Another embodiment of the present invention is a method to determine whether a breast cancer patient should have aggressive treatment and follow up after initial diagnosis of breast cancer, the method consisting of determining that patient's signature status. If the patient is positive, that patient is considered to be at high risk for metastasis and therefore needing more aggressive treatment and follow up, to prevent or identify metastasis. If the patient signature-negative that patient is considered to be at a low risk for metastasis.
  • the signature can include one or more of the following: miR-335, -206 and -126 and the genes collagen, type I, alpha 1 (COLl Al), receptor-type tyrosine protein phosphatase (PTPRN), c- Mer tyrosine kinase (MERTK), phospholipase C beta 1 (PLCBl), tenascin C (TNC) and SRY-box containing transcription factor (SOX4).
  • Preferred targets for the diagnosis include
  • Another embodiment of the present invention is a method to provide to a breast cancer patient a prognosis of metastasis-free survival consisting of obtaining breast tumor cells, or breast tumor tissue, from that breast cancer patient, determining the RNA or gene expression levels on those breast tumor cells, or breast tumor tissue, determining an expression score based on the expression of these targets and considering patients whose expression scores are below the mean expression level determined from a random sample of primary breast cancer cells or breast cancer tissue to have worse metastasis-free survival prognosis than those patients that are above such mean expression level.
  • the targets can include one or more of the following: miR-335, -206 and -126 and the genes collagen, type I, alpha 1 (COLl Al), receptor-type tyrosine protein phosphatase (PTPRN), c-Mer tyrosine kinase (MERTK), phospholipase C beta 1 (PLCBl), tenascin C (TNC) and SRY-box containing transcription factor (SOX4).
  • Preferred targets for the diagnosis include SOX4, TNC, and miR-335
  • the present invention also teaches a method of treating metastatic breast cancer.
  • the metastatic breast cancer may be determined using the above techniques, or any other technique.
  • the treatment is done by administering oligonucleotides that will increase the levels of underexpressed miRNA or decrease the level of over expressed genes.
  • oligonucleotides include: antisense oligonucleotides, ribozymes, shRNA, miRNA, siNA, and dsRNA.
  • the amount given is sufficient to cause a change in the expression of the target.
  • RNA RNA molecule(s), RNA segment(s) and RNA fragment(s) may be used interchangeably to refer to RNA that mediates RNA interference. These terms include double-stranded RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA).
  • Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Collectively, all such altered RNAi compounds are referred to as analogs or analogs of naturally-occurring RNA.
  • RNA of the present invention need only be sufficiently similar to natural R]SfA that it has the ability to mediate RNAi.
  • the phrase "mediate RNAi" refers to and indicates the ability to distinguish which mRNA are to be affected by the RNAi machinery or process. RNA that mediates RNAi interacts with the RNAi machinery such that it directs the machinery to degrade particular mRNAs or to otherwise reduce the expression of the target protein.
  • the present invention relates to RNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or lack of expression of the target mRNA.
  • RNA interference or "RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811, incorporated herein by reference). dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi involves mRNA degradation, but many of the biochemical mechanisms underlying this interference are unknown. The use of RNAi has been further described in Carthew et al. (2001) Current Opinions in Cell Biology 13, 244-248, and Elbashir et al.
  • RNAi molecules of the invention are double-stranded or single-stranded RNA of from about 21 to about 23 nucleotides which mediate RNA inhibition. That is, the isolated RNAi of the present invention mediate degradation of mRNA of the hsp27 gene.
  • siRNA can be introduced directly into a cell to mediate RNAi (Elbashir et al., 2001, Nature 411 :494 498). Many methods have been developed to make siRNA, e.g, chemical synthesis or in vitro transcription. Once made, the siRNAs are introduced into cells via transient transfection.
  • siRNA expression vectors have also been developed to continually express siRNAs in transiently and stably transfected mammalian cells (Brummelkamp et al., 2002 Science 296:550 553; Sui et al., 2002, PNAS 99(6):5515 5520; Paul et al., 2002, Nature Biotechnol. 20:505 508). Some of these vectors have been engineered to express shRNAs, which get processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing.
  • Another type of siRNA expression vector encodes the sense and antisense siRNA strands under control of separate pol III promoters (Miyagishi and Taira, 2002, Nature Biotechnol. 20:497 500). The siRNA strands from this vector, like the shRNAs of the other vectors, have 3' thymidine termination signals. Silencing efficacy by both types of expression vectors was comparable to that induced by transiently transfecting siRNA.
  • Antisense technology is also useful for reducing expression of a gene.
  • DNA DNA, RNA or a chemical analogue
  • mRNA messenger RNA
  • the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA.
  • Antisense oligonucleotides act by blocking translation or causing degradation of mRNA.
  • This synthesized nucleic acid is termed an "anti-sense” oligonucleotide because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called the “sense” sequence (so that a sense segment of mRNA 5'-AAGGUC-3' would be blocked by the anti-sense RNA segment 3'-UUCCAG-5' ).
  • mRNA messenger RNA
  • the present invention may be used to introduce oligonucleotides into a cell for the treatment or prevention of disease.
  • dsRNA may be introduced into a cancerous cell or tumor and thereby inhibit gene expression of a gene required for maintenance of the carcinogenic/ tumorigenic/ metastatic phenotype.
  • a target gene may be selected which is required for initiation or maintenance of the disease/pathology. Treatment would include amelioration of any symptom associated with the disease or clinical indication associated with the pathology.
  • a vector encoding a nucleic acid molecule of the invention can be administered to a cell.
  • Viral vectors include, without limitation, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a vaccinia virus, a herpes viruses, and a bovine papilloma virus.
  • a nucleic acid molecule of the invention or a vector encoding such a nucleic acid can be encapsulated in, for example, a nanoparticle or a liposome, and administered to a cell.
  • the present invention also concerns methods and reagents useful in reducing metastatic characteristics of cancer cells therefore providing a therapy useful in the treatment of cancer.
  • the present invention introduces RNA or DNA able to express RNA into a cell for the treatment or prevention of breast cancer.
  • dsRNA may be introduced into a cancerous cell or tumor and thereby inhibit gene expression of a gene required for maintenance of the carcinogenic/tumorigenic phenotype.
  • a target gene may be selected which is required for initiation or maintenance of the disease/pathology. Treatment would include amelioration of any symptom associated with the disease or clinical indication associated with the pathology.
  • the present invention also relates to genes that are overexpressed in certain breast cancers, making the breast cancer more prone to growth and metastatic behavior. Such genes can be targeted individually or in groups for higher therapeutic effect.
  • Another embodiment of the present invention is a method of treating breast cancer consisting of providing to a patient in need of such treatment small nucleic acid molecules, or a plasmid able to drive the expression of such small nucleic acid molecules, such as siNA, siRNA, dsRNA, miRNA, or shRNA capable of mediating RNAi that contain the sequence Seq. ID No. 1.
  • small nucleic acid molecules such as siNA, siRNA, dsRNA, miRNA, or shRNA capable of mediating RNAi that contain the sequence Seq. ID No. 1.
  • Another embodiment of the present invention is a method of treating breast cancer consisting of providing to a patient in need of such treatment small nucleic acid molecules, or a plasmid able to drive the expression of such small nucleic acid molecules, such as siNA, siRNA, dsRNA, miRNA, shRNA capable of mediating RNAi that contains the sequence Seq. ID No. 2.
  • small nucleic acid molecules such as siNA, siRNA, dsRNA, miRNA, shRNA capable of mediating RNAi that contains the sequence Seq. ID No. 2.
  • a method of treating breast cancer consisting of providing to a patient in need of such treatment small nucleic acid molecules, or a plasmid able to drive the expression of such small nucleic acid molecules, such as siNA, siRNA, dsRNA, miRNA, shRNA molecules capable of mediating RNAi that contain the sequence Seq. ID No. 3.
  • small nucleic acid molecules such as siNA, siRNA, dsRNA, miRNA, shRNA molecules capable of mediating RNAi that contain the sequence Seq. ID No. 3.
  • Another embodiment of the present invention is a method of treating breast cancer consisting of providing to a patient in need of such treatment an oligonucleotide comprising Seq. ID No. 21 or Seq. ID No. 22.
  • the present invention also discloses compositions that are useful in treating metastasizing breast cancer.
  • This includes oligonucleotides capable of regulating expression of genes and miRNA that are abnormally expressed in metastases. These oligonucleotides maybe: antisense oligonucleotides, ribozymes, shRNA, miRNA, siNA, and dsRNA. It is the aim of the present invention to provide molecules that can be useful in the treatment of cancer, specifically breast cancer. Moreover, the molecules provided in this invention are also useful in limiting and/or eliminating the metastatic potential of breast cancer cells.
  • Molecules useful in the practice of the present invention are molecules that can mediate RNAi of one or more of the following genes: collagen, type I, alpha 1 (COLlAl), receptor-type tyrosine protein phosphatase (PTPRN), c-Mer receptor tyrosine kinase (MERTK), phospholipase C beta 1 (PLCBl), tenascin C (TNC) and SRY-box containing transcription factor (SOX4).
  • Such molecules contain a sequence of at least 20 nucleotides that is at least 90% identically, but ideally 100% identical, to the complementary sequence of the RNA that is being targeted, ie the RNA of one or more of those genes.
  • oligonucleotide sequence Once such an oligonucleotide sequence has been identified, it can be used to create a variety of siNA, siRNA, dsRNA, miRNA, shRNA molecules capable of mediating RNAi. All such molecules contain the short oligonucleotide sequence or a sequence at least 90% identical to it.
  • nucleotide molecules, and the corresponding protein product in the case of genes, that are either increased or decreased in breast cancer provides an opportunity for therapy of such cancer.
  • genes that are overexpressed in such cancers with respect to normal cells or genes that are overexpressed in certain breast cancer types that have bad prognosis with respect to breast cancer types that have a good prognosis provide an avenue for targeted therapy.
  • Such targeted therapy can be implemented using small molecules in the case of protein products that have enzymatic functions that can be targeted through the use of such small molecules.
  • targeted inhibition of gene expression may also be implemented via the use of polynucleotide compounds, such as but not limited to, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, where the nucleotide sequence of such compounds is related to the RNA sequence that is intended to be targeted.
  • Antisense technology has been the most commonly described approach in protocols to achieve sequence-specific interference. For antisense strategies, stoichiometric amounts of single-stranded nucleic acid perfectly or partially complementary to the RNA of interest are introduced into the cell.
  • Another method for engineered interference is based on a triple helical nucleic acid structure. This approach relies on the ability of certain nucleic acid populations to adopt a triple-stranded structure.
  • the present invention relates to types of molecules identified for targeted therapy of breast cancer.
  • One is a series of RNA molecules that are naturally found in a human cell and that control the expression, through RNAi, of a series of genes. Such molecules have been found to be at low levels in certain breast cancers. It is the object of the present invention to provide a therapy that can increase the level of those molecules in breast cancer cells so that the expression of the genes they control is lowered so that the phenotype of such breast cancer cell becomes less malignant and its metastatic ability is either eliminated or reduced.
  • RNA molecules are of the type known as microRNAs.
  • RNAi short hairpin RNA
  • shRNA short hairpin RNA
  • This invention describes those microRNAs and their shRNA sequence.
  • the same RNAi can be accomplished through the use of other RNA types as long as a core sequence of around 21 nucleotides is present in a double stranded form, with perfect or partially imperfect sequence complementarity in that double strand.
  • the oligonucleotide may comprise one or more strands of polymerized ribonucleotide. It may include modifications to either the phosphate-sugar backbone or the nucleoside. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in oligonucleotide structure may be tailored to allow specific genetic inhibition while avoiding a general panic response in some organisms which is generated by dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase, oligonucleotides maybe produced enzymatically or by partial/total organic synthesis. Any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
  • Oligonucleotides containing a nucleotide sequences identical to a portion of the target gene are preferred for inhibition as this provides greater specificity and controls for non- desirable targeting.
  • oligonucleotides sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see http://www.genelink.com/sirna/RNAicustomorder.asp; Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory oligonucleotides and the portion of the target gene is preferred.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 5O.degree. C. or 7O.degree. C. hybridization for 12 16 hours; followed by washing).
  • the length of the identical nucleotide sequences maybe at least 25, 50, 100, 200, 300 or 400 bases.
  • One hundred percent sequence identity between the oligonucleotides and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • Oligonucleotides may be synthesized either in vivo or in vitro. Endogenous polymerase of the cell may mediate transcription in vivo, or cloned polymerase can be used for transcription in vivo or in vitro.
  • a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
  • a regulatory region may be used to transcribe the oligonucleotides strand (or strands).
  • Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.
  • the oligonucleotide strands may or may not be polyadenylated; the oligonucleotide strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
  • Oligonucleotides may be chemically or enzymatically synthesized by manual or automated reactions.
  • the oligonucleotides maybe synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6).
  • the oligonucleotides may be purified prior to introduction into the cell.
  • RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof.
  • the oligonucleotides may be used with no or a minimum of purification to avoid losses due to sample processing.
  • the oligonucleotides may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
  • Oligonucleotides maybe directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing an organism in a solution containing the oligonucleotides. Physical methods of introducing nucleic acids, for example, injection directly into the cell or extracellular injection into the organism, may also be used. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the oligonucleotides may be introduced.
  • an RNAi molecule is used to decrease gene expression of genes related to metastasis. Since the only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded mRNA, the cell has enzymes that recognize and cut dsRNA into fragments containing 21-25 base pairs (approximately two turns of a double helix and which are referred to as siRNA). The antisense strand of the fragment separates enough from the sense strand so that it hybridizes with the complementary sense sequence on a molecule of endogenous cellular mRNA. This hybridization triggers cutting of the mRNA in the double-stranded region, thus destroying its ability to be translated into a polypeptide. Introducing dsRNA corresponding to a particular gene thus knocks out the cell's own expression of that gene in particular tissues and/or at a chosen time.
  • siRNAs of the present invention can be generated by any method known in the art, for example, by in vitro transcription, recombinantly, or by synthetic means (e.g., having either a TT or a UU overhang at the 3' end).
  • siRNAs can be generated in vitro by using a recombinant enzyme, such as T7 RNA polymerase, and DNA oligonucleotide templates, or can be prepared in vivo, for example, in cultured cells ⁇ See, e.g., Elbashir et al., supra; Brummelkamp et al., supra; and Lee et al., 2002, Nat. Biotech., 20:500).
  • DNA constructs or plasmids containing DNA versions of such siRNA, dsRNA, miRNA or shRNA downstream of adequate promoters can be made so that, once such constructs are inside the cell, such RNAi molecules are continuously produced.
  • One method to produce such constructs is described in Harper SQ, Davidson BL. Methods MoI Biol. 2005;309:219-35, but there are many other methods that are known in the art.
  • strategies have been described for producing a hairpin siRNA from vectors containing a RNA polymerase III promoter.
  • Various vectors have been constructed for generating hairpin siRNAs in host cells using either an Hl-RNA or an snU6 RNA promoter.
  • RNA molecule as described above e.g., a first portion, a linking sequence, and a second portion
  • RNA polymerase III When transcribed by RNA polymerase III, the first and second portions form a duplexed stem of a hairpin and the linking sequence forms a loop.
  • the pSuper vector (OligoEngines Ltd., Seattle, Wash.) also can be used to generate siRNA.
  • a TT TT T penta-nucleotide usually is attached to the end of the second portion (i.e., the antisense strand) in a vector to serve as a terminator for RNA polymerase III transcription. For that reason, siRNA candidates that contain more than three consecutive Ts should be avoided since four or more consecutive Ts in the template nucleic acid triggers termination of RNA polymerase III transcription.
  • siRNAs There are well-established criteria for designing siRNAs ⁇ See, e.g., Elbashire et al., 2001, Nature, 411:494 8; Amarzguioui et al., 2004, Biochem. Biophys. Res. Commun., 316(4): 1050 8; Reynolds et al., 2004, Nat. Biotech., 22(3):326-30). Details can be found in the websites of several commercial vendors such as Ambion, Dharmacon, GenScript, and OligoEngine. Other such algorithm can be found at http://www.genelink.com/sirna/RNAicustomorder.asp.
  • any potential siRNA candidate generally is checked for any possible matches to other nucleic acid sequences or polymorphisms of nucleic acid sequence using the BLAST alignment program ⁇ See ncbi.nlm.nih.gov on the World Wide Web). Typically, a few iRNAs have to be generated and screened in order to compare their effectiveness.
  • Double-stranded (ds) RNA can be used to interfere with gene expression in mammals.
  • dsRNA is used as RNAi of the function of a nucleic acid molecule of the invention to produce a phenotype that is the same as that of a null mutant of a nucleic acid molecule of the invention (Wianny & Zernicka-Goetz, 2000, Nature Cell Biology 2: 70 75).
  • the double- stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell.
  • RNA molecules maybe at least 10, 12, 15, 20, 21, 22, 23, 24, 25, 30, nucleotides in length.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • the therapeutic agent may be introduced directly into the cancer of interest via direct injection.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion or inhalation), transdermal (topical), transmucosal, and rectal administration.
  • siRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • solvents dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • Solutions or suspensions can include the following components: a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)), fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), glycerine, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)),
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged administration of the injectable compositions can be brought about by including an agent that delays absorption. Such agents include, for example, aluminum monostearate and gelatin.
  • the parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic.
  • Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be liquid, or can be enclosed in gelatin capsules or compressed into tablets. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of an oral composition.
  • Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as macrocrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dosage unit forms of the invention are dependent upon the amount of a compound necessary to inhibit proliferation of the cancer cells.
  • the amount of a compound necessary to inhibit proliferation of the cancer cells can be formulated in a single dose, or can be formulated in multiple dosage units. Treatment may require a one-time dose, or may require repeated doses.
  • oligonucleotides that can be used in the present invention are short interfering nucleic acid (siNA), siRNA, double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNAi that contain the sequence of Seq. ID Nos. 1, 2, or 3.
  • Other specific sequences are plasmid constructs that drive the expression of a small nucleic acid molecule, such as short interfering nucleic acid (siNA), siRNA, double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) capable of mediating RNAi that contain the sequence: Seq. ID No. 1, Seq. ID No. 2, or Seq. ID No. 3.
  • the present the invention also relates to small nucleic acid molecules, such assiNA, siRNA, dsRNA, miRNA, or shRNA capable of mediating RNAi against any of COLlA, PTPRN, MERKT, PLCBl, TNC and SOX4 RNAs useful for the treatment of breast cancer.
  • small nucleic acid molecules such as assiNA, siRNA, dsRNA, miRNA, or shRNA capable of mediating RNAi against any of COLlA, PTPRN, MERKT, PLCBl, TNC and SOX4 RNAs useful for the treatment of breast cancer.
  • Another embodiment of the present invention is a plasmid construct containing a DNA sequence of the shRNA molecule capable of mediating RNAi against Tenascin C, the shRNA containing the sequence: Seq. ID No. 4 or Seq. ID No. 5.
  • Antisense has been shown to regulate Tenascin C in pulmonary hypertension using Seq. ID No. 21 and Seq. ID No. 22.
  • Cowan, K. et al, Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression, of vascular disease, J. Clin. Invest. 2000 January 1; 105(1): 21-34.
  • RNAi interference with sequences target the first 164 bases starting from the 5'-end of TNC has been demonstrated by Zukiel et al, Cancer biology & therapy 2006: VoL 5, pp. 1002-1007; "Suppression of human brain tumor with interference RNA specific for tenascin-C" which is incorporated herein by reference.
  • siNA molecule or a plasmid construct able to drive the expression of such siNA, capable of mediating RNAi against Tenascin C, the siRNA containing the sequence: Seq. ID No. 6.
  • siNA molecule or a plasmid construct able to drive the expression of such siNA, capable of mediating RNAi against Tenascin C, the siRNA containing the sequence: Seq. ID No. 7.
  • oligonucleotides capable of reducing expression of COLlAl are known from Millington-Ward et al, Eur. J Human Genetics (2004) 12, 864-866, and Wang et al, Experimental dermatology 2007- JuI; vol 16 (issue 7) : pp 611-7, which are incorporated herien by reference.
  • oligoncleotides capable of reducing expression of SOX4 are known from Pramoonjago et al, . Oncogene (2006) 25, 5626-5639, which is incorporated herein by reference.
  • the diagnostic assay described above can be suitably practiced using just one of the markers (genes or miRNA) and provide an acceptable result. However, it may be desirable to perform tests on a sample for more than one of the markers. This is facilitated by a kit that includes at least the sequence specific reagents for the detection and quantification of two or more of the markers, i.e.
  • SRY-box containing transcription factor 4 SOX4
  • TTC Tenascin C
  • PPRN receptor-type tyrosine protein phosphatase
  • collagen type I, alpha 1 (COLl Al)
  • MERTK Mer receptor tyrosine kinase
  • PLCBl phospholipase C beta 1
  • suitable reagents include antibodies, oligonucleotide probes, polymerases and reverse transcriptase.
  • the kit may also include buffers, enzymes, membranes and gels suitable for the assays.
  • kits include reagents for detection of S 0X4, TNC or miR335 as one of the reagent types.
  • Other embodiments of kits include reagents for detection of SOX4 and TNC, or SOX4, TNC and or miR335.
  • Standard values for the control signature (mean and standard deviation) for a detected/quantified material in non-metastatic breast cells may be included in the kit, for example as a printed insert.
  • Hierarchical clustering based on the expression of these miRNAs correctly classified the MDA-MB-231 derivatives into three groups comprising the BoMl lines, the LM2 lines and the parental lines, respectively.
  • Hierarchical clustering on the basis of the expression of the 20 miRNAs for which expression was most altered across various cell lines again correctly classified the MDA-MB-231 derivatives into these three groups.
  • the most salient finding was a set of eight miRNAs for which expression was decreased across all metastatic sub-lines compared with their expression in the parental line.
  • qRT-PCR quantitative stem-loop polymerase chain reaction
  • Figs. IA-D show systematic identification of miRNAs that suppress lung and bone metastasis in multiple human breast cancer cell derivatives.
  • Fig. IA shows Bioluminescence imaging of lung metastasis by lung metastatic breast cancer cells with restored expression of specific miRNAs. 1 ' 104 LM2 cells expressing individual miRNAs or the control hairpin, as well as the parental MDA-MB-231 cells, were inoculated intravenously into immunodeficient mice. Lung colonization was measured by bioluminescence and quantified
  • Figs. 2A-C shows miR-126 suppresses overall tumour growth and proliferation whereas miR-335 and miR-206 regulate migration and morphology.
  • Fig. 7 shows quantification of miRNA expression in LM2 cells with restored expression of miRNAs.
  • Fig. 8 shows bioluminescence quantification of lung colonization by additional miRNAs that are downregulated in metastatic cells.
  • LM2 cells were transduced with a retro virus encoding a control hairpin or precursors of endogenous human miRNAs. 1x104
  • mice To determine whether the expression of these miRNAs is lost in other human breast cancer cells with enhanced metastatic propensity, we inoculated mice with a purified population of malignant cells (CN34) obtained from the pleural fluid of a patient with metastatic breast cancer who was treated at our institution. Gupta, Nature 446, 765. We then isolated human tumour cells from metastatic lesions that formed in the lungs (CN34-LM1 cells) and bones (CN34-BoMl cells) of the mice. Both the CN34-LM1 and CN34-BoMl derivatives displayed a loss of miR-335, miR-126 and miR-206 expression relative to the parental population from which they were isolated (Fig. 9C).
  • miR-335, miR-206 and miR- 126 significantly reduced the ability of CN34-LM1 and CN34-BoMl cells to metastasize to lung (Fig. 1C) and bone (Fig. ID), respectively.
  • the expression of these miRNAs therefore, is lost in multiple, independently derived, breast cancer samples and their restoration suppresses lung and bone metastasis in these distinct metastatic cell populations.
  • Fig. 9A-C shows loss of metastasis suppressor miRNA expression from primary human cancer cells as well as exogenously introduced vector subsequent to metastatic progression.
  • Fig. 9 A Quantification of the loss of metastasis miRNA expression from transduced vector upon in vivo metastatic selection.
  • 1X104 LM2 cells expressing metastasis suppressor miRNAs (miR-335, miR-206, and miR-126) were inoculated intravenously into immunodeficient mice. The lungs of mice that emitted a bioluminescence signal were extracted at 10 weeks and rare solitary nodules were identified through ex- vivo bioluminescence and extracted.
  • n 4-6; Error bars indicate s.e.m. p-values based on a one-sided rank-sum test.
  • n 3; Error bars indicate s.e.m. p-values determined using a one-tailed student's t-test.
  • LM2 cells and CN34-BoMl cells expressing miR-335 or miR-206 as a population contained a significantly lower fraction of elongated cells than did control cells (Fig. 12).
  • LM2 cells expressing these miRNAs continued to express the mesenchymal marker vimentin, in vivo, suggesting that the suppression of metastasis in these cells was not due to classical mesenchymal-epithelial transition.
  • LM2 cells were transduced with individual miRNAs or the control hairpin, cells were inoculated into mammary fat pads of immunodeflcient mice. The mammary fat pad tumours were subsequently resected, and sections were immunohistochemically stained in automated fashion for the mesenchymal marker vimentin. We postulated that such an alteration in shape may be associated with a decrease in cell motility, which would limit metastatic migration. Indeed, in trans- well migration assays, LM2 cells and CN34-BoMl cells expressing miR-335 or miR-206 displayed a significant reduction in migration compared with controls (Fig. 2C).
  • miR-335 and miR-206 also caused a significant reduction in invasive capacity, as assessed through matrigel invasion assays (Fig. 13). These findings highlight cell-autonomous mechanisms of cell proliferation, migration and invasion through which specific miRNAs may suppress metastasis.
  • Figs. 1 IA-B show immunohistochemical analysis for apoptotic and proliferation markers in mammary tumours.
  • Fig. 1 IA - Quantification of phospho-histone 3 through immunohistochemical staining of mammary fat pad tumours; n 6; error bars indicate s.e.m.; p-values were calculated using a one-sided rank-sum test.
  • Fig. 1 IB - Quantification of apoptosis through immunohistochemical staining of mammary fat pad tumours for cleaved caspase-3; n 6; error bars indicate s.e.m.; p-values were calculated using a one-sided rank- sum test.
  • Fig. 1 IA - Quantification of phospho-histone 3 through immunohistochemical staining of mammary fat pad tumours; n 6; error bars indicate s.e.m.; p-values were calculated using a one-sided rank- sum test.
  • the CN-BoMl and its derivatives were in general more elongated than the MDA-MB-231 derivatives, therefore cells with a length:width ratio greater than 5:2 were considered elongated and are plotted as a fraction of the total cells for each CNBoMl line.
  • n 195-300 cells for MDA-MB-231 derivative lines and 171-174 cells for CN34 lines; Error bars represent s.e.m. p-values obtained using a one-sided rank sum test comparing fraction of cells per field.
  • Fig. 13 shows miR-335 and miR-206 suppress invasion by lung metastatic cells.
  • tumours comprised large (>2.5 cm) and small ( ⁇ 2.5 cm) oestrogen receptor (ER)-positive and -negative subtypes that were surgically resected before administration of chemotherapy.
  • This set consisted of primary tumours resected from 11 patients that ultimately relapsed to lung, bone, or brain as well as tumours resected from nine patients who did not suffer metastatic relapse (Table 1).
  • qRT-PCR (Fig. 14) revealed that patients whose primary tumours displayed low expression of miR-335, miR-126 (Fig. 3A) or miR-206 (Fig. 15) had a shorter median time to metastatic relapse.
  • the median expression values of these miRNAs was more than eightfold lower in tumours from patients that ultimately relapsed compared to tumours from patients that did not relapse (Fig. 14).
  • Fig. 14 shows quantification of miRNA levels in human breast tumour samples.
  • Each vertical bar represents expression levels of metastasis suppressor miRNAs miR-335, miR- 206, and miR-126 in addition to miR-122a and miR-199a.
  • the expression level of a given miRNA in each sample was normalized to the median expression value for that miRNA across the 20 tumour samples.
  • the Y-axis is represented in Iog2 scale. Vertical black bars correspond to tumours extracted from patients that subsequently suffered metastatic relapse.
  • Fig. 15 shows the association between the loss of miR-206 expression in primary breast tumours and metastatic relapse, a, miR-206 expression was assessed in a set of twenty primary breast tumour specimens through qRT-PCR.
  • Kaplan-Meier curve depict metastasis-free survival of patients whose primary tumours contained low or high levels of the miR-206.
  • n 20; p-value obtained using a log-rank test.
  • Example 5 miR-335 regulates a set of metastasis genes
  • Bioinformatic analysis of the 3'-UTRs of these genes revealed them all to have at least six nucleotides of sequence complementarity to the miR-335 seed region (data not shown).
  • To determine whether the altered expression of these genes in metastatic cells is, in part, mediated through their 3'-UTRs we cloned the 3'-UTR of five of these genes downstream of a luciferase gene as a reporter, and assayed their expression in LM2 cells (low miR-335) and MDA-MB-231 cells (high miR-335).
  • SOX transcription factors are known to regulate progenitor cell development and migration. Hoser, J. Neurosci. 27, 5495-5505.
  • mutation of the miR-335 seed sequence in the SOX4 UTR as well as miR-335 inhibition with an antagomir significantly increased reporter expression (Fig. 4D).
  • Restoration of miR-335 expression in LM2 cells reduced endogenous SOX4 messenger RNA expression, whereas miR-335 inhibition increased SOX4 expression in MDA-MB-231 cells (Fig. 4E).
  • the knockdown of SOX4 in LM2 cells with either of two unique short hairpin RNAs (shRNAs) reduced the overall fraction of elongated cells, similar to the phenotype observed with miR-335 restoration in these cells (Fig. 16A).
  • Figs. 3A-B shows clinical association of miR-335 and miR-126 with metastasis-free survival.
  • Fig. 3 A miR-335, miR-126, miR-122a and miR-199a expression was assessed in a set of 20 primary breast tumour samples through qRT-PCR. Kaplan-Meier curves depict metastasis-free survival of patients whose primary tumours contained low or high levels of the indicated miRNAs. P-values were obtained using a log-rank test.
  • Fig. 3B The parental MDA-MB-231 cells were transfected with antagomirs targeting endogenous miR-335, miR- 199a* or a control antagomir.
  • Figs. 4A-E show a miR-335-regulated gene set includes SOX4 as a miR-335 direct target.
  • Fig. 4 A The miR-335 metastasis signature consists of genes downregulated by miR- 335 and are also overexpressed in bone as well as lung metastatic MDA-MB-231 derivatives.
  • the heatmap depicts the variance-normalized expression values for each gene averaged across two MDA-MB-231 samples, four LM2 samples and two LM2/miR-335 samples.
  • the scale bar depicts standard deviation change from the mean for the expression value of each gene.
  • Fig. 4B UTR reporter assays of miR-335 metastasis genes in LM2 and MDA-MB-231 cells.
  • Fig. 4E SOX4 expression in LM2 and MDA-
  • MB-231 cells was obtained through real time qRT-PCR in cells expressing miR-335 or transfected with the miR-335 antagomir.
  • n 3; error bars represent s.e.m.; P-values derived using a one-sided student's t-test.
  • Figs. 5A-C show miR-335 regulates metastasis and invasion through suppression of SOX4 and TNC.
  • Fig. 5 A -C show miR-335 regulates metastasis and invasion through suppression of SOX4 and TNC.
  • 5B - 2 ' 105 LM2 cells transduced with either a control short hairpin, a short hairpin targeting SOX4 or one targeting TNC were inoculated intravenously into immunodeficient mice.
  • Figs. 16A-B show the miR-335 regulated genes S OX4 and TNC alter the morphology of lung metastatic cells and suppress migration.
  • 16B - 2.5X104 LM2 cells expressing either of two short-hairpin RNAs targeting SOX4, a hairpin targeting TNC, or an siRNA targeting TNC, or a short hairpin control vector, were seeded onto trans-well inserts and migration through a trans-well membrane insert was quantified. Images of cells that had migrated through the inserts were obtained using Metamorph software and cells were counted in automated fashion using Metamorph software. n 6; p-values based on a one-sided student's t-test.
  • Figs. 17A-B show the miR-335 regulated six-gene signature predicts overall metastasis-free survival in the MSK-82 and EMC-286 datasets.
  • Fig. 17A - The MSK-82 cohort, which consists of 82 patients with primary breast cancer 18, and the EMC-286 cohort, consisting of 286 patients with primary breast cancer 27, were classified into two groups based on the expression of the miR-335 regulated signature in primary tumours. Kaplan- Meier curves representing metastasis-free survival of patients whose original tumours expressed the miR-335 signature (positive) and those whose original tumours did not (negative).
  • Fig. 17B Kaplan-Meier curves representing lung-metastasis-free survival and bone-metastasis-free survival of MSK-82 patients whose original tumours expressed the miR-335 signature, p-values based on a Mantel-Cox Log-rank test.
  • miR-335, miR-206 and miR-126 are selectively downregulated across a number of highly metastatic human cell lines compared to the general tumour cell population and have demonstrated abilities to suppress metastasis of breast cancer cells to different organ sites.
  • the expression of miR-335 and miR-126 in human mammary tumours is inversely associated with metastatic relapse of these tumours to distant organs, and the expression of miR-206 also shows a trend in the same direction.
  • the expression of a set of genes regulated by one of these miRNAs, miR-335 is directly associated with relapse. We also establish two of these genes, SOX4 and TNC, as in vivo .
  • miR-10b was identified as a miRNA whose overexpression in breast cancer cells promoted tumour growth and lung micrometastasis.
  • Ma Nature 449, 682-688.
  • MicroRNAs are thus uncovered as another class of molecules, along with metastasis suppressor genes (Steeg, P. S. Metastasis suppressors alter the signal transduction of cancer cells. Nature Rev. Cancer 3, 55-63 (2003)), that negatively regulate tumour progression.
  • miRNA-3375 regulates a set of putative, and a subset of validated, metastasis genes argues that the multi-gene regulatory capacity of a miRNA can function as a barrier to tumour progression in humans.
  • the strong association of the loss of miR-335 and miR-126 expression with metastatic relapse suggests the potential for the use of these molecules in prognostic stratification of breast cancer patients in addition to conventional clinical and pathological staging markers.
  • Gene expression analysis was performed of the LM2 cells and bone-metastatic BoM2 cells, using as reference the parental, non-metastatic, MDA-MB-231 line.
  • the gene-list below shows genes with more than two-fold increase expression in the metastatic cell lines compared to the parental, non-metastatic cell line, and was obtained through an analysis of available gene-expression datasets for MDA-MB-231 derivative lung metastatic cell lines 4175, 4180, 4142, 4173, and bone metastatic cell lines 2287 and 1833. Kang, Cancer Cell 3, 537-549; Minn, Nature 436, 518-524.
  • RNAi of SOX 4 via a SOX 4 specific shRNA, or indirectly through overexpression of miR-335 induced a phenotype of more elongated LM2 cells compared to the LM2 cells that were used as control, indicating that SOX4 constitutes a target gene for the treatment of breast cancer and prevention or reduction of metastatic potential of that cancer.
  • RNAi against SOX4 and TNC was performed using either specific shRNA or specific siRNA
  • Fig 16B shows that reduction of SOX4 or TNC via RNAi significantly reduced the ability of the LM2 cells to migrate in a trans-well migration assay, which is a proxy for metastatic ability.
  • the MDA-MB-231 cell line and its metastatic derivatives (Kang, Cancer Cell 3, 537- 549; Minn, Nature 436, 518-524; Minn, A. J. et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J. Clin. Invest. 115, 44-55 (2005)) as well as the pleural effusion-derived CN34 cancer cells have been described previously. Gomis, R. R., Alarcon, C, Nadal, C, Van Poznak, C. & Massague, J. C/EBP at the core of the TGF cytostatic response and its evasion in metastatic breast cancer cells. Cancer Cell 10, 203-214 (2006).
  • Voorhoeve, Cell 124, 1169-1181 consist of approximately 500-bp fragments that span a given miRNA genomic region under the control of a CMV promoter. Voorhoeve, Cell 124, 1169-1181; Chen, Science 303, 83-86. All animal work was done in accordance with the MSKCC Institutional Animal Care and Use Committee. Statistical analyses were performed using the Graphpad Prism 5 software package.
  • MDA-MB-231 line and its metastatic derivatives have been described previously. Minn, Nature 436, 518-524; Minn, J. Clin. Invest. 115, 44-55 (2005). All cell lines were cultured in DMEM supplemented with 10% FBS, glutamine, pyruvate, penicillin, streptomycin, and fungizone. CN34 cancer cells were isolated from the malignant pleural effusion of a patient with metastatic breast cancer treated at our institution upon obtaining written consent following IRB regulations as previously described. Gomis, Cancer Cell 10, 203-214 (2006).
  • the GPG29 retroviral packaging cell line was maintained in DMEM containing 10% FBS supplemented with G418, puromycin, penicillin, streptomycin, doxycycline, and fungizone, as previously described. Minn, Nature 436, 518-524. AU transfections were done using Lipofectamine 2000 (Invitrogen). Subsequent to transfection, GPG29 cells were maintained in DMEM supplemented with 10% FBS and pyruvate.
  • RNA from MDA-MB-231 cells, its derivatives, or CN34 cells and its derivatives was extracted and purified using the MiRvana kit (Ambion). Tissues from primary breast cancers were obtained from therapeutic procedures performed as part of routine clinical management at our institution. Methods for primary tumour sample processing were described previously. Minn, Nature 436, 518-524. hi brief, RNA from these samples was extracted from frozen tissues by homogenization in TRIzol reagent (Gibco/BRL), as described previously. Minn, Nature 436, 518-524. Taqman MicroRNA assays (Applied Biosystems) were used to quantify mature miRNA expression as previously described. Chen, Nucleic Acids Res. 33, el79 (2005).
  • RNU6 (Applied Biosystems) was used as endogenous control for miRNA expression studies.
  • each reverse-transcriptase (RT) reaction consisted of 50 ng of purified total RNA, IX RT buffer, dNTPs (each at 0.375 mM), 5 U ⁇ l-1 MultiScribe reverse transcriptase, 5OnM stem-loop RT primer, and 0.38 U ⁇ l-1 RNase inhibitor (Applied Biosystems). RT reactions were incubated at 16 0 C for 30 min, 42 0 C for 30 min, and 85 0 C for 5 min. Real-time PCR reactions for miRNAs from cell lines were performed in quadruplicate in lO ⁇ l volumes.
  • the Real-time reaction mix consisted of 0.67 ⁇ l of RT product, 0.5 ⁇ l of 2OX TaqMan microRNA assay mix, 5 ⁇ l TaqMan 2X universal PCR Master Mix. Quantitative miRNA expression data were acquired and analyzed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems).
  • each RT reaction contained 10 ng of purified total RNA.
  • RT-PCR reactions from archived tumour RNA were performed in replicates of six.
  • mRNA expression quantification four-hundred nanograms of total purified RNA was reverse transcribed using the Superscript III first-strand synthesis system (Invitrogen).
  • CDNA corresponding to approximately 5 ng of initial RNA was used for each quadruplicate quantitative PCR reaction.
  • Human SOX4 and ⁇ -glucuronidase (as endogenous control) were amplified using commercially designed Taqman gene expression assays (Applied Biosystems) and the Taqman Universal PCR master mix (Applied Biosystems).
  • RNA microarray hybridization 20 ⁇ g total RNA obtained from MDA-MB-231 and its derivatives was labeled and hybridized on miRNA microarrays by LC sciences (Houston, TX). The arrays were designed to detect miRNA transcripts corresponding to 453 miRNAs contained in the Sanger miRBase Release 8.2. Of all the probes assayed, those corresponding to 179 miRNAs revealed a signal above a threshold above background in at least one of the MDA-MB-231 cell lines. Raw signal intensities representing hybridization to probes were mean-normalized across across cell lines.
  • Hierarchical clustering of variance- normalized expression values was performed using Matlab software. Hierarchical clustering was performed using euclidean distance as a metric and utilizing average linkage. Clustering of cell types along organotropic and metastatic phenotypes was achieved using both the expression of 179 miRNAs as well as the 20 miRNAs that displayed the highest coefficients of variation across cell lines.
  • RNA microarray hybridization and miR-335 signature The gene expression datasets from MDA-MB-231 cell derivatives as well as the primary breast tumour gene expression datasets from the MSK and EMC cohorts have been published previously. Minn, Nature 436, 518-524; Wang, Lancet 365, 671-679 (2005). Transcriptional profiling of LM2 cells expressing miR-335 was performed as previously described (Minn, Nature 436, 518- 524) through the use of the Affymetrix HG-Ul 33 plus 2.0 array by the genomics core laboratory at MSKCC. 874 probes corresponding to 756 genes displayed a greater than 0.2 loglO (ratio) fold decrease in expression relative to LM2 cells expressing a short hairpin control.
  • This low permissive threshold was chosen so as to include genes whose fold reduction at the transcript level as detected by hybridization may be much less than their potential reduction at the protein level.
  • the number of transcripts downregulated at the permissive threshold as well as the number of transcripts downregulated at the less- permissive threshold of two-fold (458 probes) is comparable to profiling analyses with other miRNAs. He, L. et al. A microRNA component of the p53 tumour suppressor network. Nature 447, 1130-1134 (2007).
  • the miR-335 regulated 6-gene signature consists of the set of genes corresponding to the 756 miR-335 downregulated genes that 1) were also upregulated by more than two-fold in both lung and bone metastatic MDA-MB-231 derivatives relative to the parental line (available gene expression data-set Minn, Nature 436, 518-524) and 2) were expressed by multiple MDA-MB-231 cell lines. Generation of retrovirus, miRNA expressing cells, and knockdown cells. The plasmid vectors encoding the miRNAs were kindly provided by R. Agami (Voorhoeve, Cell 124, 1169-1181), and have been previously described.
  • these PMSCV-based vectors were individually transfected into the GPG29 amphotropic packaging cell line. Virus particles were collected at 48 and 72 h after transfection, filtered, and concentrated through ultracentrifugation, as previously described. Gupta, Nature 446, 765. These concentrated retroviral particles were then used to transduce cells in the presence of 8 ⁇ g ml-1 polybrene. Vector-transduced cells were selected with blasticidin. miRNA expression was confirmed in all cases using quantitative RT-PCR for the mature forms of individual miRNAs, suggesting that the precursor transcripts were endogenously processed to the mature forms.
  • miRNA expression did not interfere with endogenous processing of miRNAs as revealed by qRT- PCR for the expression of other miRNAs in these cell lines.
  • MDA-MB-231 or LM2 cells were transfected with antagomirs (Meister, RNA 10, 544-550) (25 ⁇ M; Ambion) based on manufacturer's recommendations.
  • antagomirs Meister, RNA 10, 544-550
  • MDA-MB-231 cells were transiently transfected with the control anti-miR Locked Nucleic Acid (LNA)- 159, LNA-335, or LNA-199a* (2OnM Exiqon) based on manufacturers recommendations.
  • LNA anti-miR Locked Nucleic Acid
  • Transfected cells were inoculated into immunodeficient mice via tail-vein injection four days post transfection.
  • SOX4 knockdown cells were generated through infection with Mission TRC lentiviral particles (Sigma- Aldrich) containing short hairpins targeting the human SOX4 gene.
  • the TRC numbers and the shRNA sequences that yielded the greatest knockdown were TRCN0000018216: Seq. ID No. 8 and TRCNOOOOO 18217: Seq. ID No. 9. These sequences contain complementary portions set forth in seq. ID Nos. 19 and 20, respectively.
  • TNC knockdown cells were generated either by infection with a pRetrosuper vector expressing the forward sequence Seq. ID No. 4 or by transfecting cells with the following ON-TARGET siRNA with sense sequence: Seq. ID No. 6 (Dharmacon).
  • UTR reporter assays The full-length 3' UTRs of the miR-335 regulated human genes COLlAl, SOX4, PTPRN2, TNC, MERTK, as well as the control gene UBE2F were amplified from human genomic DNA (Novagen) and individually cloned into the Psicheck 2 dual luciferase reporter vector (Promega). MDA-MB-231 or LM2 cells were then transfected with each reporter construct and MDA-MB-231 cells were also transfected with a miR-335 antagomir (25 nM) targeting endogenous miR-335 (ambion).
  • miR-335 antagomir 25 nM
  • NCI NOD/SCID female mice
  • Athymic female mice (NCI) age-matched between 6- 8 weeks were used for bone metastasis xenograft assays.
  • 5X105 viable cells were re-suspended in a 1 :1 mixture of PBS and growth-factor-reduced Matrigel (BD Biosciences) and injected orthotopically into the fourth mammary gland in a total volume of 25 ⁇ l.
  • 2X104 viable cells were re- suspended in 0.1 ml PBS and introduced into the arterial circulation via intra-cardiac injection, as previously described.
  • Kang Cancer Cell 3, 537-549. Bone metastatic colonization was monitored and quantified using non-invasive bioluminescence of normalized hind-limb photon flux at Day 35.
  • Trans-well migration and invasion assays Cancer cells were conditioned overnight in 0.2% FBS ECM media without growth factors. The next day, cells were pulsed with 5 ⁇ M cell tracker green (Invitrogen) for 30 minutes, trypsinized, and re-suspended in 0.2% FBS ECM media without growth factors. Tumour cells were then seeded at 25000 cells per well into trans-well inserts (3 ⁇ pore size, BD Falcon) for migration assays or at 50000 cells per well into matrigel-coated, growth factor reduced, invasion chambers (8 ⁇ pore size, BD Biosciences). The wells were washed with PBS and fixed with 4% paraformaldehyde after 6 hours for migration assays or after 22 hours for invasion assays.
  • the Mann- Whitney test was used to determine the significance of difference between the expression levels for miRNAs in tumours that ultimately relapsed relative to those that did not.
  • the correlation of miR-335 or miR-126 expression status with ER histologic or Her-2/Neu amplification status was determined by calculating the significance of the Spearman correlation coefficients in a two- tailed test using Graphpad Prism 5 software.
  • Primary tumour gene expression data from the MSK (Minn, Nature 436, 518-524) and EMC (Wang, Lancet 365, 671-679 (2005)) cohorts were used to classify each tumour as miR-335 signature positive if the sum of Z-scores for the expression values of the 6 miR-335 regulated genes was greater than one standard deviation from the mean.
  • Previously published metastasis-free survival data corresponding to these tumours was then plotted in Kaplan-Meier form for patients whose tumours were miR-335 signature positive versus negative.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Selon la présente invention, six gènes qui sont surexprimés dans le cancer du sein métastasique, et en particulier dans des métastases osseuses et pulmonaires du cancer du sein, et dont la surexpression est également corrélée à une sous-expression de miR-335, miR-206 and/or miR-126, ont été identifiés. L'évaluation des taux d'un ou plusieurs des produits génétiques ou des miARN constitue un indicateur pronostique/diagnostique de la probabilité de la présence de métastases du cancer du sein. De plus, des agents qui diminuent la quantité de produit génétique protéique et/ou augmentent la quantité d'un ou plusieurs des miARN associés peuvent être utilisés dans le traitement du cancer du sein métastasique.
PCT/US2008/088065 2007-12-22 2008-12-22 Pronostic et traitement véhiculé par interférence du cancer du sein Ceased WO2009082744A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US864307P 2007-12-22 2007-12-22
US61/008,643 2007-12-22
US1075708P 2008-01-10 2008-01-10
US61/010,757 2008-01-10

Publications (2)

Publication Number Publication Date
WO2009082744A2 true WO2009082744A2 (fr) 2009-07-02
WO2009082744A3 WO2009082744A3 (fr) 2010-01-14

Family

ID=40801748

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/088065 Ceased WO2009082744A2 (fr) 2007-12-22 2008-12-22 Pronostic et traitement véhiculé par interférence du cancer du sein

Country Status (1)

Country Link
WO (1) WO2009082744A2 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2327781A1 (fr) * 2009-11-27 2011-06-01 RWTH Aachen Micro ARN et réparation de tissu
WO2014066498A1 (fr) * 2012-10-23 2014-05-01 Vivek Mittal Traitement du cancer du sein métastatique
EP2828387A1 (fr) * 2012-03-21 2015-01-28 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Nouvelle cible thérapeutique pour la prévention de la métastase de tumeurs
US8980261B2 (en) 2011-02-11 2015-03-17 The Rockefeller University Treatment of angiogenesis disorders
CN106566838A (zh) * 2016-11-14 2017-04-19 上海伯豪生物技术有限公司 一种基于CRISPR‑Cas9技术的miR‑126全长基因敲除试剂盒及其应用
CN113151468A (zh) * 2014-06-13 2021-07-23 东丽株式会社 乳癌的检测试剂盒或装置以及检测方法
KR20210092356A (ko) * 2020-01-15 2021-07-26 주식회사 베르티스 암의 진단용 조성물
CN113667748A (zh) * 2021-07-19 2021-11-19 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） circIKBKB的抑制剂及其检测试剂在乳腺癌骨转移诊断、治疗和预后试剂盒的应用
CN115490768A (zh) * 2021-06-18 2022-12-20 佛山热休生物技术有限公司 Col1a1的表位肽及所述表位肽与热休克蛋白的复合物
CN116196397A (zh) * 2023-02-09 2023-06-02 山西锦波生物医药股份有限公司 重组i型人源化胶原蛋白在乳腺癌治疗中的用途
WO2023224499A3 (fr) * 2022-05-17 2024-02-15 Medicofarma Biotech Sa Molécule d'arnsi contre la ténascine-c humaine (tnc) et composition pharmaceutique la comprenant
CN117737246A (zh) * 2024-02-01 2024-03-22 首都医科大学附属北京天坛医院 Plcb1基因作为生物标志物在侵袭性垂体腺瘤中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030065156A1 (en) * 1997-12-23 2003-04-03 Williams Lewis T. Novel human genes and gene expression products I
EP1838870A2 (fr) * 2004-12-29 2007-10-03 Exiqon A/S Nouvelles compositions d'oligonucleotides et sequences de sondes utiles pour la detection et l'analyse de microarn et de leurs marn cibles
EP1969147B1 (fr) * 2006-01-05 2014-07-30 The Ohio State University Research Foundation Méthodes à base de microARN pour le diagnostic de cancer de l'estomac

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2327781A1 (fr) * 2009-11-27 2011-06-01 RWTH Aachen Micro ARN et réparation de tissu
WO2011064354A3 (fr) * 2009-11-27 2011-09-29 Rwth Aachen Micro-arn et réparation tissulaire
US10894989B2 (en) 2011-02-11 2021-01-19 The Rockefeller University Treatment of angiogenesis disorders
EP3301176A1 (fr) 2011-02-11 2018-04-04 The Rockefeller University Procédé d'identification d'acides nucléiques régulant la métastasation
US8980261B2 (en) 2011-02-11 2015-03-17 The Rockefeller University Treatment of angiogenesis disorders
EP2673363A4 (fr) * 2011-02-11 2015-04-15 Univ Rockefeller Traitement de troubles d'angiogenèse
US9493841B2 (en) 2011-02-11 2016-11-15 The Rockefeller University Treatment of angiogenesis disorders
US12060619B2 (en) 2011-02-11 2024-08-13 The Rockefeller University Treatment of angiogenesis disorders
EP3604534A1 (fr) 2011-02-11 2020-02-05 The Rockefeller University Procédé d'identification d'acides nucléiques de régulation de la métastatisation
EP2828387A1 (fr) * 2012-03-21 2015-01-28 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Nouvelle cible thérapeutique pour la prévention de la métastase de tumeurs
US9512425B2 (en) 2012-10-23 2016-12-06 Cornell University Inhibiting migration of cancer cells
WO2014066498A1 (fr) * 2012-10-23 2014-05-01 Vivek Mittal Traitement du cancer du sein métastatique
US12398430B2 (en) 2014-06-13 2025-08-26 Toray Industries, Inc. Breast cancer detection kit or device, and detection method
CN113151468A (zh) * 2014-06-13 2021-07-23 东丽株式会社 乳癌的检测试剂盒或装置以及检测方法
CN106566838B (zh) * 2016-11-14 2019-11-01 上海伯豪生物技术有限公司 一种基于CRISPR-Cas9技术的miR-126全长基因敲除试剂盒及其应用
CN106566838A (zh) * 2016-11-14 2017-04-19 上海伯豪生物技术有限公司 一种基于CRISPR‑Cas9技术的miR‑126全长基因敲除试剂盒及其应用
KR102325742B1 (ko) * 2020-01-15 2021-11-15 주식회사 베르티스 암의 진단용 조성물
KR20210092356A (ko) * 2020-01-15 2021-07-26 주식회사 베르티스 암의 진단용 조성물
CN115490768A (zh) * 2021-06-18 2022-12-20 佛山热休生物技术有限公司 Col1a1的表位肽及所述表位肽与热休克蛋白的复合物
CN113667748A (zh) * 2021-07-19 2021-11-19 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） circIKBKB的抑制剂及其检测试剂在乳腺癌骨转移诊断、治疗和预后试剂盒的应用
WO2023224499A3 (fr) * 2022-05-17 2024-02-15 Medicofarma Biotech Sa Molécule d'arnsi contre la ténascine-c humaine (tnc) et composition pharmaceutique la comprenant
CN116196397A (zh) * 2023-02-09 2023-06-02 山西锦波生物医药股份有限公司 重组i型人源化胶原蛋白在乳腺癌治疗中的用途
CN116196397B (zh) * 2023-02-09 2025-07-04 山西锦波生物医药股份有限公司 重组i型人源化胶原蛋白在乳腺癌治疗中的用途
CN117737246A (zh) * 2024-02-01 2024-03-22 首都医科大学附属北京天坛医院 Plcb1基因作为生物标志物在侵袭性垂体腺瘤中的应用
CN117737246B (zh) * 2024-02-01 2025-01-28 首都医科大学附属北京天坛医院 Plcb1基因作为生物标志物在侵袭性垂体腺瘤中的应用

Also Published As

Publication number Publication date
WO2009082744A3 (fr) 2010-01-14

Similar Documents

Publication Publication Date Title
WO2009082744A2 (fr) Pronostic et traitement véhiculé par interférence du cancer du sein
Tavazoie et al. Endogenous human microRNAs that suppress breast cancer metastasis
Zhang et al. Identification of the tumor‑suppressive function of circular RNA FOXO3 in non‑small cell lung cancer through sponging miR‑155
Zhang et al. miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer
Ge et al. Long noncoding RNA CAR10 promotes lung adenocarcinoma metastasis via miR-203/30/SNAI axis
Zhang et al. microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN
Tie et al. MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor
Li et al. Epigenetic silencing of miR-338-3p contributes to tumorigenicity in gastric cancer by targeting SSX2IP
US9085804B2 (en) Ultraconserved regions encoding ncRNAs
Kojima et al. MicroRNA122 is a key regulator of α-fetoprotein expression and influences the aggressiveness of hepatocellular carcinoma
Jones et al. miRNA signatures associate with pathogenesis and progression of osteosarcoma
Huang et al. The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis
Saito et al. MicroRNA-196a is a putative diagnostic biomarker and therapeutic target for laryngeal cancer
Suh et al. FHIT suppresses epithelial-mesenchymal transition (EMT) and metastasis in lung cancer through modulation of microRNAs
Zhan et al. MicroRNA-548j functions as a metastasis promoter in human breast cancer by targeting Tensin1
Zhi et al. Circular RNA profiling identifies circ102049 as a key regulator of colorectal liver metastasis
JP2019512489A (ja) マイクロｒｎａおよびその使用方法
JP2012500389A (ja) 多発性脊髄腫の診断、予後および治療のためのマイクロｒｎａに基づく組成物および方法
JP2011511634A (ja) ガン幹細胞に関連する方法および組成物
Foruzandeh et al. CircRNAs as potent biomarkers in ovarian cancer: a systematic scoping review
Song et al. Deregulated expression of miR-224 and its target gene: CD59 predicts outcome of diffuse large B-cell lymphoma patients treated with R-CHOP
Zhang et al. RETRACTED ARTICLE: KDM5C Expedites Lung Cancer Growth and Metastasis Through Epigenetic Regulation of MicroRNA-133a
US20120108655A1 (en) Methods of Diagnosing and Treating Carcinomas
JP6193269B2 (ja) ｍｉＲ−１９９ａ−５ｐ、その標的及び／又は阻害剤の、線維増殖障害の診断、予後予測及び治療のための使用
WO2024222591A1 (fr) Utilisation d'un inhibiteur de l'expression de l'arnlnc loc646762 et médicament

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08863425

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08863425

Country of ref document: EP

Kind code of ref document: A2