WO2013144325A1 - Enzymes d'élongation d'acide gras en tant que cibles pour le diagnostic et la thérapeutique du cancer - Google Patents

Enzymes d'élongation d'acide gras en tant que cibles pour le diagnostic et la thérapeutique du cancer Download PDF

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WO2013144325A1
WO2013144325A1 PCT/EP2013/056790 EP2013056790W WO2013144325A1 WO 2013144325 A1 WO2013144325 A1 WO 2013144325A1 EP 2013056790 W EP2013056790 W EP 2013056790W WO 2013144325 A1 WO2013144325 A1 WO 2013144325A1
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expression
biological sample
cells
elovl2
genes
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Johannes SWINNEN
Muralidhara Rao BAGADI
Jelle Machiels
Johannes BERKERS
Natalie Rueda RINCON
Jonas DEHAIRS
Eyra MARIEN
Evelyne LERUT
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Katholieke Universiteit Leuven
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Priority claimed from GBGB1205486.2A external-priority patent/GB201205486D0/en
Priority claimed from GBGB1205520.8A external-priority patent/GB201205520D0/en
Application filed by Katholieke Universiteit Leuven filed Critical Katholieke Universiteit Leuven
Publication of WO2013144325A1 publication Critical patent/WO2013144325A1/fr

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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
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/60—Growth-hormone releasing factors (GH-RF) (Somatoliberin)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)
    • G01N2333/91057—Acyltransferases other than aminoacyltransferases (general) (2.3.1) with definite EC number (2.3.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7057—(Intracellular) signaling and trafficking pathways
    • G01N2800/7066—Metabolic pathways
    • G01N2800/7085—Lipogenesis or lipolysis, e.g. fatty acid metabolism

Definitions

  • Methods and compositions are provided for the diagnosis and treatment of cancers, for example, renal cell carcinoma, prostate cancer, gastrointestinal, and colorectal cancer.
  • the methods and compositions are related to the fatty acyl chain elongation phenotypes observed in various tumors, such as the fatty acyl chain elongation mediated by the genes of the elongation pathway.
  • Phospholipids the main building blocks of membranes, can respond to cellular signals by changing the length of their fatty acyl chains and/or changing the saturation of the bonds in the chains. These changes, in turn, locally modulate the biochemical and biophysical properties of the cell membranes. Through this direct mechanism, among others, phospholipid metabolism regulates the cellular processes taking place at the membrane.
  • fatty acyl chain elongation and enzymes involved in the process may be targeted for diagnosis, treatment and prevention of cancer.
  • fatty acids particularly fatty acids with 12 or more carbons
  • the pathway for fatty acid elongation involves at least four enzymes and fatty acyl CoA, malonyl CoA, and NADPH as substrates (Fig. 2A).
  • a 3-keto acyl- CoA synthase catalyzes the condensation of malonyl CoA with a fatty acyl-CoA precursor.
  • a 3-keto acyl-CoA reductase reduces the resulting 3-keto intermediate.
  • 3- hydroxy acyl-CoA dehydratase catalyzes the dehydration of the 3-hydroxy species.
  • the product of the dehydration step is reduced in a reaction catalyzed by trans-2,3-enoyl-CoA reductase (TECR)
  • TECR trans-2,3-enoyl-CoA reductase
  • the ELOVLs elongate different fatty acids.
  • ELOVL1, ELOVL3, and ELOVL6 elongate saturated and monounsaturated fatty acids
  • ELOVL 2, ELOVL 4, and ELOVL5 elongate polyunsaturated fatty acids.
  • Other enzymes in the elongation pathway have been also identified.
  • enzymes from the acetyl-CoA carboxylase (ACC or ACAC) family catalyze carboxylation of acetyl-CoA.
  • acyl-CoA reductases belong to the hydroxysteroid dehydrogenase subfamily, for example, HSD17.
  • the family of dehydratases include PTPLs (protein tyrosine phosphatase-like members), such as PTPLA, PTPLB, PTPLAD1, and PTPLAD2.
  • Fatty acid synthase (FAS, encoded by the FASN gene) catalyzes fatty acid synthesis, in particular, the synthesis of palmitate from acetyl-CoA and malonyl- CoA, into long chain saturated fatty acids.
  • FADS1, FADS2 and SCD1 are involved in fatty acid desaturation and often work in conjunction with the elongation pathway.
  • SREBPlc is a transcription factor regulating several of the genes mentioned above.
  • ELOVL5 was identified within a panel of 27 possible markers for prostate cancer (Romanuik et al., Novel biomarkers for prostate cancer including noncoding transcripts, Am J Pathol. 2009, 175(6): 2264-2276), while the role of ELOVL7 was studied in prostate cancer after its identification in a genome wide gene expression study (Tamura et al., Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism, Cancer Res. 2009, 69(20): 8133-8140).
  • ELOVL1 and ELOVL2 have each been implicated in progression of breast cancer (Hilvo et al., Novel theranostic opportunities offered by characterization of altered membrane lipid metabolism in breast cancer progression. Cancer Res. 2011, 71(9): 3236-45 and Tanaka et al., Preventing/Remedies for Cancer, U.S. Patent Application No. 2006/0189518).
  • no study has definitively and comprehensively identified the changes in enzymatic cascades involved in fatty acyl chain elongation observed in cancer, nor has expression or role of specific changes in enzymes in cancer development been established for different types and subtypes of cancer.
  • One aspect of the present disclosure relates to a method for identifying cancer cells in a biological sample, comprising: (a) obtaining a biological sample from a patient; (b) measuring expression of one or more genes involved in fatty acid chain elongation, wherein the genes are selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc; and (c) comparing the expression of the genes with a reference gene in the same sample or biological sample with expression of the genes in a reference sample; wherein a difference between the expression of the genes in the biological sample and in the reference sample indicates that the patient sample contains cancer cells.
  • the expression of one or more genes is increased in the patient sample as compared with the reference gene or the reference sample. In certain embodiments, the expression of one or more genes is decreased in the biological sample as compared with the reference sample. The increase in expression of the genes may be concurrent with the decrease in expression of other genes. In some embodiments, the increase in expression of genes is independent of a decrease in expression of other genes.
  • Cancer cells may be characterized by an elongation phenotype.
  • the elongation phenotype comprises an increase in the presence of certain phospholipid species and/or an increase in phospholipid species with long fatty acyl chains.
  • An elongation phenotype may comprise a relative increase in longer chain phospholipid species as compared to shorter chain phospholipid species within at least one head group class.
  • the cancer cells are renal cancer cells, prostate cancer cells, or gastrointestinal cancer cells., or colorectal cancer cells.
  • renal cancer cells are renal cell carcinoma cells, for example, clear cell renal cell carcinoma cells.
  • the cancer cells are gastrointestinal cancer cells.
  • the cancer cells may be p53 positive or negative.
  • the expression of one or more genes selected from MLYCD, ELOVL1, ELOVL2, and ELOVL7 is increased in the biological sample as compared with the reference sample.
  • the expression of one or more genes selected from HSD17B12, PTPLA, and PTPLAD1 are decreased in the biological sample as compared with the reference sample.
  • the renal cell carcinoma cells are VH L negative cells. In VHL negative cells, the expression of one or more genes selected from MLYCD, ACC, ELOVL1, ELOVL2, ELOVL3, ELOVL5, ELOVL6, PTPLA, PTPLB, PTPLAD1, and TECR may be increased in the biological sample as compared with the reference sample.
  • the expression of one or more genes selected from ELOVL7 and PTPLAD2 may be decreased in the biological sample as compared with the reference sample.
  • a method for identifying cancer cells in a biological sample comprises (a) obtaining a biological sample from a patient; (b) measuring expression of ELOVL2; and (c) comparing the expression of ELOVL2 in the biological sample with expression of ELOVL2 in a reference sample; wherein a difference between the expression of the genes in the biological sample and in the reference sample indicates that the biological sample contains cancer cells.
  • the method comprises measuring in the biological sample expression of one or more additional genes selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc.
  • the additional genes may be selected from MLYCD, ELOVL1, ELOVL7, HSD17B12, PTPLA, and PTPLAD1.
  • the expression of one or more genes may be increased in the biological sample as compared with the reference sample, and/or may be decreased in the biological sample as compared with the reference sample.
  • the biological sample may comprise prostate cancer cells, foe example adenocarcinoma cells.
  • the prostate cancer cells for example the adenocarcinoma cells, may be castration-resistant prostate cancer cells.
  • Prostate cancer cells may express the oncogenic fusion gene TM PRSS2-ERG.
  • prostate cancer cells may express the androgen receptor.
  • the expression of one or more genes selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc is increased in the biological sample as compared with the reference sample.
  • the expression of one or more genes selected from M LYCD, ELOVL3, ELOVL4, ELOVL6, ELOVL7, and PTPLAD2 is decreased in the biological sample as compared with the reference sample.
  • the biological sample is a cellular sample.
  • the biological sample may also be a membrane vesicle.
  • the biological sample is an exosome.
  • cancer cells as identified and analyzed herein are characterized by an elongation phenotype.
  • a further aspect of the present disclosure relates to an inhibitor of one or more of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2, TECR, FADS1, FADS2, SCD1, SCD5, ACLY, and SREBPlc for use in treating cancer.
  • the may be specific for ELOVL2.
  • the inhibitor reduces cancer cell proliferation.
  • FIGURES Figure 1 shows a heatmap and clustering of relative changes in phospholipid species in ccRCC tumor tissue versus matched normal tissue from 20 ccRCC patients.
  • Phospholipids are ordered according to lipid class (PC, PE, PS and PI). Within each PL class, species are ranked according to the degree of unsaturation and within each subclass of (un)saturation according to their chain length..
  • Figure 2 shows (A) the metabolic pathways of fatty acyl chain elongation; (B) differential expression of enzymes in tumor versus normal tissue in renal cell carcinoma patients; (C) impact of VH L status on acyl chain elongation; (D) regulation of these enzymes by VH L; (E) impact of VHL on ELOVL2 expression.
  • Figure 3 shows the effect of VH L and ELOVL2 knockdown on accumulation of lipid droplets in RCC4 cells.
  • Figure 4 shows that the inhibition of lipid elongation by soraphen decreases proliferation in RCC4 VH L negative cells.
  • Figure 5 shows that ELOVL2 expression modifies ciliogenesis, cyst formation and podocalyxin expression in M DCK cells.
  • Figure 6 shows a heatmap showing relative changes in phosphatidylcholine and sphingomyelin species in urinary exosomes from 7 kidney cancer patients before versus after surgical tumor resection. Green squares indicate a decrease in PL species in urinary exosomes before surgery compared to urinary exosomes after surgery, while red squares represent an increase as indicated by the scale bar (log2). Cluster analysis divided patients in several subgroups.
  • Figure 7 shows a heatmap showing relative changes in phosphatidylserine species in urinary exosomes from 9 kidney cancer patients before versus after surgical tumor resection.
  • Green squares indicate a decrease in PL species in urinary exosomes before surgery compared to urinary exosomes after surgery, while red squares represent an increase as indicated by the scale bar (log2).
  • Figure 8 shows relative changes in phopholipid species in (A) urinary and (B) plasma exosomes from a cancer patient versus a healthy control.
  • Figure 9 shows androgen regulation of lipid metabolizing enzymes in two prostate cancer cell lines treated with two concentrations of androgens. Enzyme expression was measured by qPCR.
  • Figure 10 shows effects of ELOVL6 inhibition on elongation of PC, PE, PS and PI lipid species in HCT116 colon cancer carcinoma cells.
  • FIG 11 shows effects of ELOVL6 inhibition on phosphorylation of EGFR in HCT116 cells.
  • HCT116 cells were treated with Elovl6 inhibitor ( ⁇ ) for 72 hours.
  • Cells were stimulated with EGF (lOng/ml) for 2 minutes and lysed in IX sample buffer.
  • Cell lysates were analyzed by WB for EGFR and pEGFR tyr 1068.
  • Figure 12 shows effects of ELOVL6 inhibition on migration of HCT116 cells.
  • HCT116 cells were treated with Elovl6 inhibitor ( ⁇ ) for 72h or DMSO as a control.
  • Cells were seeded in Boyden chambers using 10% FBS with or without HGF (lOng/ml) and EGF (lOng/ml) as chemoattractants. After 48h migrated cells were stained with crystal violet and counted (Y-axis, number of migrated cells xlOO).
  • Figure 13 shows the relative abundance and changes in relative abundance (Log 2 ) of 4 lipid species: (A) PC, (B) PE, (C) PI and (D) PS in tumor cells after tyrosine kinase was inhibited in cancer cells using imatinib.
  • Figure 14 shows the changes in relative abundance (Log2) of the lipid species PC in renal cancer cells after tyrosine kinase was inhibited using sunitimib.
  • Figure 15 shows the changes in relative abundance (Log2) of the lipid species PE in renal cancer cells after tyrosine kinase was inhibited using sunitimib.
  • the present disclosure relates to enzymes involved in fatty acyl chain elongation and alterations in the expression of these enzymes in cancer cells as compared with normal healthy tissues.
  • Some cancers are characterized by a shift in the phospholipid population towards species with long acyl chains (PCT/EP2011/066568).
  • one aspect of the present disclosure is a method for identifying the phospholipid species in specific cancers.
  • the cancers are cancers with an elongation phenotype (i.e., the phospholipid species comprises a relative increase in longer chain phospholipid species as compared to shorter chain phospholipid species within at least one head group class) as described herein.
  • Another aspect is a method for identifying specific enzymes which regulate fatty acyl chain elongation in cancer.
  • a further aspect relates to identification and diagnosis of cancer, on the basis of the expression of the enzymes and/or the genes encoding the enzymes.
  • a method for diagnosing and/or identifying cancer cells in a patient sample comprises (a) obtaining a biological sample from a patient, (b) measuring expression of one or more genes involved in fatty acid chain elongation, wherein the genes are selected from FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc; and (c) comparing the expression of the genes in the biological sample with a reference gene within the same sample or with the expression of the genes in a reference sample; wherein a difference between the expression of the genes in the biological sample and in the reference sample indicates that the biological sample
  • the change in gene expression may be an increase in expression of one or more of the genes and/or a decrease in expression of one or more of the genes.
  • the change in gene expression may be measured as an increase (or decrease) in expression of the gene and/or the gene product, for example, a nucleic acid or protein encoded by the gene.
  • the identification of a cancer type or subtype is based on a profile in which the expression of specific genes are increased, while the expression of other genes is decreased.
  • the biological sample is obtained from the tissue and/or bodily fluid of a patient.
  • the reference sample may be also obtained from a patient, for example, the reference sample may be healthy tissue from the same patient whose biological sample is analyzed.
  • the reference sample may also comprise other cancer tissue.
  • the cancer is characterized by an increase in the presence of certain phospholipid species and/or an increase in phospholipid species with long fatty acyl chains.
  • the extent to which a cancer is invasive and/or metastatic correlates with the presence of phospholipid species and expression of enzymes which regulate fatty acyl chain elongation.
  • the expression profile of one or more of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc guides the development of novel treatments, for example, in a companion diagnostic setting and/or guides the selection of patients for clinical trials.
  • biomarkers associated with specific cancer types or subtypes As cancer treatment shift towards personalized and targeted treatments for individual patients, it is essential to utilize biomarkers associated with specific cancer types or subtypes. This way, a patient whose cancer expresses certain biomarkers can be diagnosed and treated with the appropriate therapies.
  • monitoring the expression of biomarkers on cancer before, during, and after treatment may provide information about the treatment response, toxicity, and other side effects, even before clinical manifestations emerge.
  • the profiles of phospholipids and/or elongation enzyme as described herein are used to identify specific cancer types and subtypes in patients, predict disease prognosis, and determine response to treatment.
  • the expression of one or more of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc may be increased or decreased in a sample from a patient, as compared with a reference sample.
  • the expression profile of these enzymes may then be used to diagnose the type and/or subtype of cancer and determine a suitable treatment regimen. Moreover, before, during, and after the treatment, the expression of one or more of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc may be measured. Treatment could be modified according to the change in expression of the enzymes. For example, if the expression profile indicated that the cancer was not responding to treatment, then an alternative treatment could be used.
  • lipid elongation profile is altered in cancer cells after treatment with a TKI, as compared to the lipid elongation profile in the cancer cells before treatment.
  • the lipid elongation profile and/or the expression profile of one or more of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc may be used to predict the response of a cancer to a TKI, select an appropriate TKI, monitor the response of a cancer to the TKI, and/or determine toxicity of the TKI. This approach may allow practitioners to avoid the use of inappropriate TKI drug therapy in patients and avoid unnecessary morbidity.
  • biological samples are obtained from a patient and comprise cells, subcellular organelles, vesicles, exosomes, bodily fluids, and/or a combination of these.
  • a biological sample may be a biological sample such as a biopsy taken from a patient.
  • a biological sample may comprise subcellular components.
  • the biological sample comprises membrane vesicles or portions of a cell membrane, and/or comprises exosomes.
  • a biological sample may also be a bodily fluid from a patient, or may be a subfraction isolated from the biological sample or bodily fluid.
  • FASN AAAA
  • ACC ACACA
  • MLYCD ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc
  • a biological sample or in vesicles such as exosomes.
  • the cancer cells are renal cancer cells, prostate cancer cells, gastrointestinal cancer cells, or colorectal cancer cells.
  • renal cancer cells are renal cell carcinoma cells, for example, clear cell renal cell carcinoma cells.
  • the cancer cells are gastrointestinal stromal cells or colorectal cancer cells.
  • the genes in the reference sample are identical to the genes measured in the biological sample.
  • the reference sample is healthy tissue. Healthy tissue should not comprise the cancer cells which will be identified by the methods described herein. Healthy tissue may be obtained from the same patient or from a different patient.
  • the genes in the reference sample are not identical to the genes measured in the biological sample. For example, if the reference sample is the same as the biological sample, then one or more reference genes measured in the reference sample would be different from the genes measured in the biological sample.
  • biomarkers associated with specific cancer types or subtypes As cancer treatment shift towards personalized and targeted treatments for individual patients, it is essential to utilize biomarkers associated with specific cancer types or subtypes. This way, a patient whose cancer expresses certain biomarkers can be diagnosed and treated with the appropriate therapies.
  • monitoring the expression of biomarkers on cancer before, during, and after treatment may provide information about the treatment response, toxicity, and other side effects, even before clinical manifestations emerge.
  • the profiles of phospholipids and/or elongation enzyme as described herein are used to identify specific cancer types and subtypes in patients, predict disease prognosis, and determine response to treatment.
  • the expression of one or more of FASN, ACC (ACACA), MLYCD, ELOVLl, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc may be increased or decreased in a biological sample from a patient, as compared with a reference sample.
  • the expression profile of these enzymes may then be used to diagnose the type and/or subtype of cancer and determine a suitable treatment regimen. Moreover, before, during, and after the treatment, the expression of one or more of FASN, ACC (ACACA), MLYCD, ELOVLl, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc. Treatment could be modified according to the change in expression of the enzymes. For example, if the expression profile indicated that the cancer was not responding to treatment, then an alternative treatment could be used.
  • Prediction of disease prognosis is a key step in treating cancer patients.
  • Expression of one or more genes selected from FASN, ACC (ACACA), MLYCD, ELOVLl, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc may be correlated with survival rates in cancer patients.
  • Renal cell carcinoma is a form of kidney cancer that originates in the lining of the proximal convoluted tubule, a structure in the kidney that filters blood and removes waste products. At least six subtypes of renal cell carcinoma have been classified, including clear cell renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, collecting duct carcinoma, and clear cell papillary renal cell carcinoma.
  • the renal cell carcinoma comprises cells that have lost function of the Von Hippel-Lindau (VHL) tumor suppressor.
  • VHL Von Hippel-Lindau
  • the biological sample obtained from a patient may comprise VHL negative cells, in contrast to the reference sample comprising VHL positive cells.
  • the VH L-negative cells may have lost at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of VH L function, as compared to VH L- positive cells.
  • Renal cell carcinomas such as VHL-negative cells and/or clear cell renal cell carcinoma may be identified on the basis of a lipid profile, for example, the presence of certain phospholipid species and/or an increase in phospholipid species with long fatty acyl chains.
  • a method for identifying a renal cell carcinoma such as clear cell renal carcinoma comprises (a) obtaining a biological sample from a patient; (b) measuring expression of intact phospholipid species in at least one head group class in the biological sample; and (c) comparing the expression of the intact phospholipid species in the biological sample with expression of the intact phospholipid species in a reference sample; wherein a difference between the expression of the intact phospholipid species in the biological sample and in the reference sample indicates that the biological sample contains renal cell carcinoma cells.
  • the phospholipid species may be phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and/or phosphatidylinositides.
  • an elongation phenotype comprises a relative increase in longer chain phospholipid species as compared to shorter chain phospholipid species within at least one head group class.
  • the shorter chain phospholipid species are phospholipid species with the same head group and equal number of unsaturations but with shorter than average (combined) acyl chain lengths, such as PC30:0, PC32:0, PC30:1, PC32:1, PC34:1, PC32:2, PC34:2, PC32:3, PC34:3, PC36:3, PC34:4, PC36:4, PC36:5, PC36:6, PC38:6; PE26:0, PE32:0, PE30:1, PE32:1, PE34:1, PE34:2, PE36:3, PE36:4, PE36:5, PE38:5, PE38:6, PE38:7, PE40:7, PE40:8, PS26:0, PS32:0, PS32:1, PS34:1, PS36:1, PS34:2, PS36:3, PS36:4, PS38:4, PS38:5, PS38:6, PS40:7; PS40:8, PI26:0, PI28:0, PI32:0, PI34:0, PI28:1, PI30:
  • an increase in at least one of the phospholipid species selected from PE40:1, PE40:3, PE40:4, PE42:6, and PI40:1, as indicated by a log2 ratio (comparing levels in the biological sample with the reference sample) > 2 indicates that the biological sample contains renal cell carcinoma cells.
  • Renal cell carcinomas such as clear cell renal cell carcinomas may also be identified on the basis of expression of genes involved in fatty acid elongation and/or on the downstream effects of these genes.
  • a method for identifying a renal cell carcinoma such as clear cell renal carcinoma comprises (a) obtaining a biological sample from a patient; (b) measuring expression of one or more genes involved in fatty acid chain elongation, wherein the genes are selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc; and (c) comparing the expression of the genes in the biological sample with expression of the genes in a reference sample; wherein a difference between the expression of the genes in the biological sample and in the reference sample indicates that the biological sample contains renal cell
  • the expression of one or more genes is increased in the biological sample as compared with the reference sample.
  • the genes are MLYCD, ELOVL1, ELOVL2, and ELOVL7.
  • the genes are MLYCD, ACC, ELOVL1, ELOVL2, ELOVL3, ELOVL5, ELOVL6, PTPLA, PTPLB, PTPLAD1, and TECR.
  • the expression of one or more genes is decreased in the biological sample as compared with the reference sample.
  • the gene is selected from HSD17B12, PTPLA, and PTPLAD1.
  • the gene is selected from ELOVL7 and PTPLAD2.
  • an increase in M LYCD, ELOVL1, ELOVL2, and ELOVL7 in combination with a decrease in HSD17B12, PTPLA, and PTPLAD1 in the biological sample as compared with the reference sample may indicate that the biological sample contains renal cell carcinoma cells, for example, clear cell renal cell carcinoma cells.
  • an increase in MLYCD, ACC, ELOVL1, ELOVL2, ELOVL3, ELOVL5, ELOVL6, PTPLA, PTPLB, PTPLAD1, and TECR in combination with a decrease in ELOVL7 and PTPLAD2 in the biological sample as compared with the reference sample may indicate that the biological sample contains renal cell carcinoma cells, for example, VHL negative cells.
  • Elongation of fatty acids may be characterized by expression of specific genes, for example in a genetic profile, or by downstream effects, such as an increase in accumulation of lipid droplets.
  • the present disclosure further relates to a method for identifying renal cell carcinoma in a biological sample, comprising (a) obtaining a biological sample from a patient; (b) measuring one or more features associated with elongation of fatty acids in the biological sample; and (c) comparing the features measured in the biological sample with features measured in a reference sample; wherein a difference between the features measured in the biological sample and in the reference sample indicates that the biological sample contains renal cell carcinoma.
  • the renal cell carcinoma is clear cell renal cell carcinoma.
  • the renal cell carcinoma comprises VHL negative cells.
  • the renal cell carcinoma may comprise a clear cell renal cell carcinoma and/or the renal cell carcinoma may comprise VH L negative cells.
  • the inhibitor may be specific for one or more genes (and/or the proteins encoded by the genes) selected from FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc.
  • the inhibitors are specific for MLYCD, ELOVL1, ELOVL2, and/or ELOVL7. In some embodiments, the inhibitors are specific for MLYCD, ACC, ELOVL1, ELOVL2, ELOVL3, ELOVL5, ELOVL6, PTPLA, PTPLB, PTPLAD1, and/or TECR; in particular MLCYD, ELOVL1, ELOVL2 and ELOVL7.
  • inhibitors may be used to reduce expression and/or function of enzymes that show increased expression in renal cell carcinoma. Exemplary inhibitors include small molecules, protein inhibitors, nucleotide inhibitors, antibodies, antisense oligonucleotides, siNAs such as siRNAs, and/or any combination of these inhibitors.
  • the inhibitor modifies renal cell carcinoma cells in the patient. Modifications may include reduced cancer cell proliferation, increased ciliogenesis, reduced cyst formation, and decreased expression of podocalyxin.
  • an ELOVL2 inhibitor may increase ciliogenesis in the renal cell carcinoma cells.
  • the ELOVL2 inhibitor reduces epithelial cyst formation in the renal cell carcinoma cells and/or reduces formation of large cysts with collapsed lumen and/or deformed cilia.
  • the ELOVL2 inhibitor reduces expression of podocalyxin, a sialoglycoprotein that is upregulated in cancers and associated with a poor prognosis.
  • the ELOVL2 inhibitor may reduce apical podocalyxin expression.
  • a further aspect of the present disclosure relates to the use of an activator for treating renal cell carcinoma in a patient in need thereof.
  • the renal cell carcinoma may comprise a clear cell renal cell carcinoma and/or the renal cell carcinoma may comprise VHL negative cells.
  • the activator may be specific for one or more genes (and/or the proteins encoded by the genes) selected from FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc.
  • the activator is specific for HSD17B12, PTPLA, and PTPLAD1. In some embodiments, the activators are specific for ELOVL7 and PTPLAD2. These activators which would be suitable for those cells in which the expression of ELOVL7 and/or PTPLAD2 is decreased, for example, VH L negative cells.
  • Activators may be used to increase expression and/or function of enzymes that show decreased expression in renal cell carcinoma. Exemplary activators include small molecules, protein activators, nucleotide activators, for example, small activating nucleic acids such as saRNA, and/or any combination of these activators. Activators may be used alone or in combination with inhibitors.
  • Prostate cancer is a common form of cancer and one of the leading causes of cancer-related mortality in men. Although many prostate tumors are slow-growing, non-metastatic, or even asymptomatic, approximately one third of all prostate cancers become aggressive and metastatic. Recurrent, metastatic prostate cancer cannot be cured. In particular, treatment is complicated by the tendency of prostate cancer cells to become refractory to medications, for example androgen receptor antagonists, approximately 1-3 years after initiating treatment.
  • prostate cancer for example, adenocarcinoma.
  • the prostate cancer is refractory to androgen therapy, and is a castration-resistant prostate cancer.
  • the prostate cancer has an oncogenic fusion gene called TMPRSS2-ERG (or TMP-ERG) in which the transcription factor Ets Related Gene (ERG) has fused with Transmembrane protease serine 2 (TMPRSS2).
  • TMPRSS2-ERG or TMP-ERG
  • ESG transcription factor Ets Related Gene
  • TMPRSS2 Transmembrane protease serine 2
  • prostate cancer may be identified on the basis of a lipid profile, for example, the presence of certain phospholipid species and/or an increase in phospholipid species with long fatty acyl chains.
  • a method for identifying prostate cancer comprises (a) obtaining a biological sample from a patient; (b) measuring expression of intact phospholipid species in at least one head group class in the biological sample; and (c) comparing the expression of the intact phospholipid species in the biological sample with expression of the intact phospholipid species in a reference sample; wherein a difference between the expression of the intact phospholipid species in the biological sample and in the reference sample indicates that the biological sample contains prostate cancer cells.
  • the phospholipid species may be phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and/or phosphatidylinositides.
  • an elongation phenotype comprises a relative increase in longer chain phospholipid species as compared to shorter chain phospholipid species within at least one head group class.
  • the shorter chain phospholipid species are phospholipid species with the same head group and equal number of unsaturations but with shorter than average (combined) acyl chain lengths, such as PC30:0, PC32:0, PC30:1, PC32:1, PC34:1, PC32:2, PC34:2, PC32:3, PC34:3, PC36:3, PC34:4, PC36:4, PC36:5, PC36:6, PC38:6; PE26:0, PE32:0, PE30:1, PE32:1, PE34:1, PE34:2, PE36:3, PE36:4, PE36:5, PE38:5, PE38:6, PE38:7, PE40:7, PE40:8, PS26:0, PS32:0, PS32:1, PS34:1, PS36:1, PS34:2, PS36:3, PS36:4, PS38:4, PS38:5, PS38:6, PS40:7; PS40:8, PI26:0, PI28:0, PI32:0, PI34:0, PI28:1, PI30:
  • Prostate cancer cells may also be identified on the basis of expression of genes involved in fatty acid elongation and/or on the downstream effects of these genes.
  • a method for identifying prostate cancer cells comprises (a) obtaining a biological sample from a patient; (b) measuring expression of one or more genes, wherein the genes are selected from the FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc; and (c) comparing the expression of the genes in the biological sample with expression of the genes in a reference sample; wherein a difference between the expression of the genes in the biological sample and in the reference sample indicates that the biological sample contains prostate cancer cells.
  • the prostate cancer cells may be adeno
  • the expression of at least one of ELOVL2, ELOVL5, ELOVL7, PTPLB, and FASN is increased in the biological sample as compared with the reference sample.
  • the expression of at least one of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc is decreased in the biological sample.
  • the prostate cancer is adenocarcinoma.
  • the inhibitor may be specific for one or more genes (and/or the proteins encoded by the genes) selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc.
  • the inhibitor is specific for one or more of FASN, ACC, ELOVL1, ELOVL2, ELOVL5, HSD17B12, PTPLA, PTPLB, and PTPLAD1.
  • Exemplary inhibitors may be selected from small molecules, protein inhibitors, nucleotide inhibitors, antibodies, antisense oligonucleotides, and siNAs such as siRNAs, and/or any combination of these inhibitors.
  • the prostate cancer is adenocarcinoma.
  • the activator may be specific for one or more genes (and/or the proteins encoded by the genes) selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc.
  • the activator is specific for one or more of MLYCD, ELOVL3, ELOVL4, ELOVL6, ELOVL7, and PTPLAD2.
  • the activators may be used to increase expression and/or function of enzymes that show decreased expression in prostate cancer.
  • Exemplary activators include small molecules, protein activators, nucleotide activators, for example small activating nucleic acids such as saRNA, and/or any combination of these activators. Activators may be used alone or in combination with inhibitors.
  • Colorectal cancer also known as colon cancer or bowel cancer
  • a small percentage of colorectal cancers results from genetic disorders, while the majority of cases occur spontaneously, particularly in an aging population.
  • the prognosis for treatment and survival is best when the cancer has not migrated from the lining of the bowel to the muscle layers and bowel walls.
  • three main tests are used to screen for colorectal cancer: fecal occult blood testing, flexible sigmoidoscopy and colonoscopy.
  • Other scanning methods and biomarker analysis may be used to diagnose colorectal cancers.
  • the colorectal cancer is adenocarcinoma.
  • the colorectal cancer may be identified on the basis of a lipid profile, for example, the presence of certain phospholipid species and/or an increase in phospholipid species with long fatty acyl chains.
  • a method for identifying colorectal cancer comprises (a) obtaining a biological sample from a patient; (b) measuring expression of intact phospholipid species in at least one head group class in the biological sample; and (c) comparing the expression of the intact phospholipid species in the biological sample with expression of the intact phospholipid species in a reference sample; wherein a difference between the expression of the intact phospholipid species in the biological sample and in the reference sample indicates that the biological sample contains colorectal cancer cells.
  • the phospholipid species may be phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and/or phosphatidylinositides.
  • an elongation phenotype comprises a relative increase in longer chain phospholipid species as compared to shorter chain phospholipid species within at least one head group class.
  • the shorter chain phospholipid species are phospholipid species with the same head group and equal number of unsaturations but with shorter than average (combined) acyl chain lengths, such as PC30:0, PC32:0, PC30:1, PC32:1, PC34:1, PC32:2, PC34:2, PC32:3, PC34:3, PC36:3, PC34:4, PC36:4, PC36:5, PC36:6, PC38:6; PE26:0, PE32:0, PE30:1, PE32:1, PE34:1, PE34:2, PE36:3, PE36:4, PE36:5, PE38:5, PE38:6, PE38:7, PE40:7, PE40:8, PS26:0, PS32:0, PS32:1, PS34:1, PS36:1, PS34:2, PS36:3, PS36:4, PS38:4, PS38:5, PS38:6, PS40:7; PS40:8, PI26:0, PI28:0, PI32:0, PI34:0, PI28:1, PI30:
  • decreased expression of at least one of the shorter chain phospholipid species in the biological sample, as compared to the reference sample, indicates that the biological sample contains colorectal cancer cells. In some embodiments, increased expression of at least one longer chain phospholipid species in the biological sample as compared to the reference sample indicates that the sample contains colorectal cancer cells.
  • Colorectal cancer cells may also be identified on the basis of expression of genes involved in fatty acid elongation and/or on the downstream effects of these genes.
  • a method for identifying colorectal cancer cells comprises (a) obtaining a biological sample from a patient; (b) measuring expression of one or more genes, wherein the genes are selected from the FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc; and (c) comparing the expression of the genes in the biological sample with expression of the genes in a reference sample; wherein a difference between the expression of the genes in the biological sample and in the reference sample indicates that the biological sample contains colorectal cancer cells.
  • ELVOL6 increased expression of ELVOL6 in the biological sample as compared with the reference sample indicates that the biological sample contains colorectal cells.
  • an inhibitor of ELOVL6, such as Compound A may be used to treat colorectal cancer.
  • a further aspect of the disclosure relates to inhibitors of one or more genes (and/or gene products) selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, which may be used to treat and/or prevent carcinoma.
  • one or more inhibitors are used to treat or prevent renal cell carcinoma, such as clear cell renal cell carcinoma; prostate cancer, such as adenocarcinoma; gastrointestinal cancer; and colorectal cancer.
  • One or more inhibitors may also be used to treat or prevent colorectal cancer.
  • Exemplary inhibitors reduce expression and/or enzymatic function of one or more genes
  • the inhibitors reduce the enzymatic function by decreasing expression of the genes and/or proteins, or by inhibition of the enzymes.
  • Inhibitors may reduce gene expression and/or enzymatic function by at least 10%, or may reduce function by 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or more.
  • Inhibitors may be small molecules, proteins such as antibodies, nucleic acids such as antisense oligonucleotide and/or siNAs like siRNAs.
  • Antibodies to the proteins encoded by the genes FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, or a fragment thereof may be any of polyclonal and monoclonal antibodies, as long as they are capable of recognizing FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, or a fragment thereof.
  • the antibodies may be produced using known methods for producing an antibody or antiserum, using FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, or a fragment thereof as an antigen.
  • the antisense oligonucleotide having a complementary or substantially complementary base sequence to the base sequence of an oligonucleotide encoding FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, or a fragment thereof can be any antisense oligonucleotide, so long as it possesses a base sequence complementary or substantially complementary to the base sequence of the oligonucleotide (e.g., DNA) of the present invention and capable of suppressing the expression of said DNA.
  • antisense DNA or antisense RNA may be used.
  • ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc may include, for example, a base sequence having at least about 70% homology, preferably at least about 80% homology, more preferably at least about 90% homology and most preferably at least about 95% homology, to the entire base sequence or to its partial base sequence (i.e., complementary strand to the DNA of the present invention), and the like.
  • exemplary antisense oligonucleotides are (a) an antisense oligonucleotide having at least about 70% homology, preferably at least about 80% homology, more preferably at least about 90% homology and most preferably at least about 95% homology, to the complementary strand of the base sequence which encodes the N-terminal region of the protein of the present invention (e.g., the base sequence around the initiation codon) in the case of antisense oligonucleotide directed to translation inhibition and (b) an antisense oligonucleotide having at least about 70% homology, preferably at least about 80% homology, more preferably at least about 90% homology and most preferably at least about 95% homology, to the complementary strand of the entire base sequence of the DNA of the present invention having intron, in the case of antisense oligonucleotide directed to RNA degradation by RNaseH, respectively.
  • Exemplary antisense oligonucleotide having at least about 70% homology,
  • RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (McSwiggen et al., U.S. Patent No. 7989612). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.
  • the process of post- transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358).
  • Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
  • dsRNAs double-stranded RNAs
  • the presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
  • Dicer a ribonuclease III enzyme referred to as Dicer.
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).
  • Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes.
  • Dicer has also been implicated in the excision of 21- and 22- nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834).
  • the RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA- induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
  • RISC RNA- induced silencing complex
  • RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215- 2218; and Hall et al., 2002, Science, 297, 2232-2237).
  • small RNA e.g., micro-RNA or miRNA
  • siNA molecules may be used to mediated gene silencing via interaction with RNA transcripts, for example, RNA transcripts of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post- transcriptional level.
  • RNA transcripts for example, RNA transcripts of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1
  • siRNA duplexes are most active when containing two 2-nucleotide 3'-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2'-deoxy or 2'-0-methyl nucleotides abolishes RNAi activity, whereas substitution of 3'-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity.
  • siNA molecules may be synthesized.
  • a target for example a region of the FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc gene has been selected.
  • the siNAs are no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; (e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure.
  • Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
  • a siNA molecule may also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.
  • Exemplary siNA molecules may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163).
  • siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.
  • HPLC high pressure liquid chromatography
  • siNA molecules are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells.
  • viral vectors can be used that provide for transient expression of siNA molecules.
  • chemically synthesizing nucleic acid molecules with modifications prevents their degradation by serum ribonucleases, which increases their potency.
  • modifications base, sugar and/or phosphate
  • Various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein.
  • Exemplary modifications that enhance their efficacy in cells, and/or remove of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements. Screening for Candidate Inhibitors
  • activity of at least one of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc is used to screen candidate compounds (or their salts) in order to identify inhibitors of the genes and/or gene products.
  • One aspect of the present disclosure provides methods for screening candidate compounds or salts for inhibiting the activity of FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc.
  • Exemplary activities of FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc include but are not limited to fatty acid elongation activity and cell growth promoting activity.
  • a method of screening a candidate compound or its salts comprises comparing (i) the fatty acid elongation activity or the cell growth promoting activity of a cell capable of producing the FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc in the absence of a test compound with (ii) the fatty acid elongation activity or the cell growth promoting activity of cells capable of producing ELOVL2 or ELOVL4 in the presence of a test compound.
  • Fatty acid elongation may be assessed by known methods, for example, by measuring the labeled amounts of liberated fatty acids in the experimental and in the control conditoins. Under control conditions, the following are mixed and reacted in an adequate buffer: (1) the extract of cells transfected with an expression vector of FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc, (2) a substrate fatty acid and (3) labeled malonyl CoA, and then the labeled fatty acid is extracted from the reaction solution.
  • FASN ACC
  • ACC ACC
  • M LYCD ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL
  • the following are mixed and reacted in an adequate buffer: (1) the extract of cells transfected with an expression vector of the protein (e.g., a gene product of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and SREBPlc) of the present invention, (2) a substrate fatty acid, (3) labeled malonyl CoA and (4) a test compound, and then the labeled fatty acid is extracted from the reaction solution.
  • an expression vector of the protein e.g., a gene product of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTP
  • Examples of the substrate fatty acids used include unsaturated fatty acids (e.g., eicosapentaenoic acid, docosapentaenoic acid, eicosatetraenoic acid, arachidonic acid, arachidonyl- coenzyme, docosatetraenoic acid, eicosatrienoic acid, mead acid, eicosatetraenoic acid, eicosatrienoic acid, di-homo-gamma-linolenic acid, tetracosateteraenoic acid, tetracosapentaenoic acid, docosapentaenoic acid, octadecenoic acid, eicosenic acid, dococenoic acid, tetracocenoic acid, octadecadienoic acid, eicosadienoic acid, etc.).
  • unsaturated fatty acids e.g., e
  • Unsaturated fatty acids having 18 to 24 carbon atoms may be used.
  • cis-5,8,11, 14,17- eicosapentaenoic acid (EPA), cis-7,10,13,16,19-docosapentaenoic acid (DPA), cis-5,8,11, 14- eicosatetraenoic acid (arachidonic acid), arachinonyl-CoA, cis-7,10,13,16-docosatetraenoic acid, cis- 5,8,11-eicosatrienoic acid, etc. may be used.
  • labeling agents used are radioactive isotopes (e.g., [ 3 H], [ 14 C], etc.), fluorescent substances [cyanine fluorescent dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Pharmacia Biotech), etc.), fluorescamine, fluorescein isothiocyanate, etc.] or the like.
  • radioactive isotopes e.g., [ 3 H], [ 14 C], etc.
  • fluorescent substances cyanine fluorescent dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Pharmacia Biotech), etc.), fluorescamine, fluorescein isothiocyanate, etc.] or the like.
  • a fatty acid elongation activity reaction solution [50 mM potassium phosphate/sodium hydroxide (pH 6.5), 5 ⁇ of Rotenone (Wako Pure Chemical Industries, Ltd.), ⁇ of CoA (Wako Pure Chemical Industries, Ltd.), 1 mM ATP, 1 mM NADPH and 1 mM magnesium chloride] containing a substrate fatty acid is prepared.
  • the reaction solution is dispensed in a 96-well plate, followed by incubation at 37°C for 1 minute.
  • test compound and the extract of cells derived from animal cell (e.g., CHO cell) line expressing FASN, ACC, MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2, orTECR at a high level as an enzyme source are added to the solution. After agitation, the mixture is incubated at 37°C for 60 minutes. Trichloroacetic acid is then added to become 4% and 1 ⁇ of 185 kBq [2- 14 C]-malonyl CoA (Amersham Bioscience) is further added thereto.
  • the mixture is agitated.
  • the reaction product is recovered onto Unifilter-96 GF/C (Packard) using Harvexter (Packard) and 20 ⁇ of microscinti-20 (Parkin Elmer) is added thereto.
  • the radioactivity e.g., 14 C
  • TopCount Packard
  • 50% inhibitory concentration IC 50
  • a compound which gives a lower IC 50 is selected as the compound inhibiting the activity (e.g., the fatty acid elongation activity) of ELOVL2 or ELOVL4.
  • Cell growth promoting activity is assayed by publicly known methods, e.g., by the colony formation assay or with its modifications, etc., followed by comparison.
  • Cells capable of producing FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc are, for example, the a host cell transformed with a vector containing the DNA encoding FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc.
  • animal cells such as COS7 cells, CHO cells, HEK293 cells, MCF-7 cells, etc., are used as the host.
  • test compound examples include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc.
  • a test compound may inhibit fatty acid elongation activity or the cell growth promoting activity by at least about 20%, at least 30%, or at least about 50%, as compared to the control cells.
  • a test compound may be selected as the compound capable of inhibiting the activity of FASN, ACC (ACACA), M LYCD, EL0VL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc.
  • a further aspect of the present disclosure relates to the use of a nucleotide encoding FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2,TECR, FADS1, FADS2, SCD1 and/or SREBPlc as a reagent for screening candidate compounds for their ability to inhibiting the expression of one or more of the genes.
  • screening comprises comparing expression of FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc in (1) a cell capable of producing the gene of interest, in the absence of a test compound, and (2) a cell capable of producing the gene of interest, incubated in the presence of a test compound.
  • the expression of the gene may be measured either by measuring protein levels or by measuring mRNA levels.
  • Protein levels may be determined by measuring the aforesaid protein present in the cell extract, etc., using an antibody capable of recognizing the protein of the present invention, in accordance with methods like western blot analysis, ELISA, etc., or their modifications. mRNA levels may be determined by methods such as Northern hybridization or PCR.
  • a test compound may inhibits gene expression of FASN, ACC (ACACA), M LYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc by at least about 20%, at least 30%, or at least about 50% in experimental cells as compared with control cells.
  • Activators of Enzymes in the Fatty Acyl Chain Elongation Pathway Activators of Enzymes in the Fatty Acyl Chain Elongation Pathway
  • a further aspect of the disclosure relates to activators of one or more genes (and/or gene products) selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc, which may be used to treat and/or prevent cancer, for example, renal cancer, prostate cancer, gastrointestinal cancer, and colorectal cancer.
  • genes selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc, which may be used
  • one or more activators are used to treat or prevent renal cell carcinoma, such as clear cell renal cell carcinoma;; prostate cancer, such as adenocarcinoma, gastrointestinal cancer, and colorectal cancer.
  • One or more activators may be used to treat or prevent colorectal cancer.
  • one or more activators are used to increase expression of genes and/or gene products whose expression is decreased in cancer.
  • activators expression and/or enzymatic function of one or more genes (and/or gene products) selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and/or SREBPlc.
  • the activators increase the enzymatic function by increasing expression of the genes and/or proteins.
  • Activators may increase gene expression and/or enzymatic function by at least 10%, or may increase function by 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or more.
  • Activators may be small molecules, proteins, nucleic acids for gene therapy, and/or small activating nucleic acids (siNAs) like saRNAs.
  • Another aspect of the present disclosure relates to the screening for compounds that act as activators.
  • the principles guiding screening for inhibitors, as described above, may be used to identify activators, although the assays may measure increases in expression of at least one gene and/or gene product selected from FASN, ACC (ACACA), MLYCD, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2JECR, FADS1, FADS2, SCD1 and SREBPlc.
  • Example 1 Changes in Phospholipid Composition and Fatty Acid Elongation in Renal Cell Carcinoma Tumor Tissue Samples Changes in phospholipid composition in ccRCC tumor tissue specimens were compared with matched normal tissue (Fig. 1). Lipid analysis of clear cell renal cell carcinoma of 20 patients. The relative abundances for different phospholipid species (PC, PE, PS and PI) were recorded by ESI- MS/MS in the MRM mode and expressed as tumor/normal ratio (log2). Clustering analysis was carried out by using an average linkage clustering algorithm of the Cluster 3.0 software. The clustering results were visualized using the Java TreeView 1.1.5. software. Comparison of lipid profiles revealed a remarkable shift of phospholipids towards species with longer acyl chains.
  • Fig. 2A shows the pathway of fatty acid elongation. Enzymes directly involved in elongation are indicated.
  • Fig. 2B shows the quantitative RT-PCR analysis of mRNA expression of genes of the elongation pathway in 11 ccRCC patients. Data were normalized for H PRT and 18S expression and expressed as the ratio (log2) of matched pairs of tumor versus normal tissue. Box plot boundaries represent the 25th and 75th percentiles, whiskers indicate the 10th and 90th percentiles. The median is indicated. *Significantly different (p ⁇ 0.05) expression in tumor versus normal tissue.
  • Fig. 2C shows the effect of VHL knockout on intact PC species in RCC4 cells.
  • Lipid extracts of RCC4 VH L positive and VHL negative cells were analyzed by ESI-MS/MS in the MRM mode. Data represent the ratio (log2) of VH L negative over VH L positive cells for different PC species. A clear increase in acyl chain elongation in RCC4 VH L negative cells was observed.
  • Fig. 2D shows the expression of mRNA in RCC4 VH L positive and VHL negative cells as analyzed by quantitative RT-PCR. Data were normalized for 18S rRNA expression. Data represent the ratio (log2) of VHL neg over VHL pos RCC4 cells. Bars represent means ⁇ standard deviation. *Significantly different (p ⁇ 0.05) expression in VHL neg versus VH L pos.
  • Fig. 2E shows the effect of VH L knockout on VH L and ELOVL2 protein expression in RCC4 cells, as measured by western blotting analysis, a-tubulin expression was used as a loading control.
  • phospholipid species in samples obtained from 20 RCC patients was compared to samples obtained from normal, healthy control patients.
  • Statistical comparisons were performed using a Wilcoxon rank sum test, Kruskal Wallis test, spearman rank correlation, indicating correlations between specific lipid species with different acyl chain lengths a clinical parameters.
  • Significant phospholipid species were as follows:
  • Tumor necrosis PE38:0, PI32:0, PC36:0, PE34:1, PI30:1 (multivariate analysis)
  • Example 2 Effects of Inhibition of Lipid Elongation by ELOVL2 Knockdown and Soraphen Treatment
  • the effects of VH L and ELOVL2 knockdown on accumulation of lipid droplets in RCC4 cells was analyzed. Lipid accumulation was evaluated by Oil Red O staining. Loss of VH L in RCC4 cells induces a marked stimulation of lipid droplet accumulation (Fig. 3).
  • siRNA-mediated knockdown of ELOVL2 in VHL negative RCC4 cells restores lipid accumulation to normal levels. Lipid elongation was inhibited by soraphen, leading to a decrease in proliferation in RCC4 VH L negative cells.
  • Fig. 4A shows the effect of soraphen treatment on intact PC species in RCC4 VH L negative cells.
  • Fig. 5A illustrates that ELOVL2 expression decreases ciliogenesis.
  • MDCK cells were transfected with a plasmid expressing ELOVL2. Cells were fixed and stained with an antibody against acetylated tubulin (red) to visualize primary cilia. Nuclei were stained with DAPI (blue). A graphic representation of the percentage of ciliated cells is shown on the right. Data represent means ⁇ SEM. Six random fields per condition were counted.
  • Fig. 5B shows that expression of ELOVL2 distorts epithelial cyst formation. ELOVL2-transfected or control MDCK cells were grown in Matrigel to form 3D epithelial cyst structures.
  • ELOVL2-transfected cells formed larger cysts with more than one or a collapsed lumen, and had no or deformed cilia.
  • Fig. 5C shows ELOVL2 expression increases apical podocalyxin expression.
  • MDCK cells were transfected with a plasmid expressing ELOVL2. Cells were fixed and stained with an antibody against the apical protein podocalyxin. Nuclei were stained with DAPI (blue).
  • Exosomes were isolated from pre-operative urine samples and post-operative samples taken from kidney cancer patients, and phospholipid species were analyzed.
  • Figure 6 is a heatmap showing relative changes in phosphatidylcholine and sphingomyelin species in urinary exosomes from 7 kidney cancer patients before versus after surgical tumor resection. Green squares indicate a decrease in PL species in urinary exosomes before surgery compared to urinary exosomes after surgery, while red squares represent an increase as indicated by the scale bar (log2). Cluster analysis divided patients in several subgroups.
  • Figure 7 shows the relative changes in phosphatidylserine species in urinary exosomes from 9 kidney cancer patients before versus after surgical tumor resection.
  • Green squares indicate a decrease in PL species in urinary exosomes before surgery compared to urinary exosomes after surgery, while red squares represent an increase as indicated by the scale bar (log2).
  • Figure 8 shows the relative changes phospholipid species in (A) urinary and (B) plasma exosomes in a cancer patient versus a normal patient.
  • Lipid metabolizing enzymes were measured in two prostate cancer cell lines (LNCaP and 22Rvl), after treatment in the absence of androgens or in the presences of androgens at either 10 " 10 M or 10 "9 M.
  • Figure 9 shows the results of qPCR measurements of FASN, ACC, MLYCD, SCD1, SCD5, FADS1, FADS2, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, PTPLAD1, PTPLB, HSD17, and TECR. Enzyme expression levels were measured by qPCR.
  • Receptor Tyrosine kinase activity was inhibited in cancer cells using imatinib and lipid profiles were measured.
  • Figure 13 shows the relative abundance and changes in relative abundance (Log2) of 4 lipid species: (A) PC, (B) PE, (C) PI and (D) PS in gastrointestinal stromal tumor (GIST) cells.
  • 4 lines of GIST cells were used: the responsive cell lines GIST Tl and GIST 882, and the unresponsive cell lines GIST488 and GIST430. Responsive cells die in response to the treatment or at least stop growing. Unresponsive cells are not or less affected.
  • the data indicate that tyrosine kinase inhibitors affect elongation in sensitive cancer cells but not in resistant cells.
  • ELOVL5, ELOVL6, ELOVL7, HSD17B12, PTPLA, PTPLB, PTPLAD1, PTPLAD2, and TECR is measured before and after administration of tyrosine kinase inhibitors.
  • ELOVL6 inhibition were assessed in the human colon carcinoma cell line HCT116.
  • cells were treated with either the ELOVL6 inhibitor Compound A or a DMSO control at a concentration of ⁇ for 72 hours and the elongation phenotype was scored.
  • Fig. 10 shows elongation data for 4 lipid species ((A) PC; (B) PE, (C) PS, and (D) PI). Inhibition of ELVOL6 led to a marked decrease in elongation of these species.
  • Lipid extracts were prepared and analyzed by ESI- MS/MS, as described below.
  • EGFR phosphorylation was determined in HCT116 cells after treatment with the ELOVL6 inhibitor Compound A.
  • Cells were treated with the inhibitor at a concentration of 1 ⁇ for 72 hours, then stimulated with EGF (10 ng/mL) for 2 minutes and lysed in IX sample buffer.
  • Cell lysates were analyzed by western blot for EGFR and pEGFR tyr 1068. As shown in Fig. 11, ELOVL6 inhibition decreased phosphorylation of EGFR in HCT116, as compared with control treated cells.
  • a migration assay was performed on HCT116 cells treated with either the ELOVL6 inhibitor (at a concentration of ⁇ for 72 hours) or DMSO as a control.
  • Cells were seeded in Boyden chambers using 10% FBS with or without HGF (lOng/mL) and EGF (lOng/mL) as chemoattractants. After 48 hours cells were scrubbed from upper surface and migrated cells were stained with crystal violet and counted. As summarized in Fig. 12, ELOVL6 inhibition decreased the migration of HCT116 cells.
  • Lipid extracts of the cellular and reference samples were made by homogenizing approximately 40mg of tissue in 800 ⁇ PBS with a Dounce homogenizer. An aliquot of ⁇ was set aside for DNA analysis. The remaining 700 ⁇ was transferred to a glass tube with Teflon liner and 900 ⁇ IN HCI:CH30H 1:8 (v/v), 800 ⁇ CHCI3 and 500 ⁇ g of the antioxidant 2,6-di-tert-butyl-4- methylphenol (BHT) (Sigma, St. Louis, MO) were added.
  • BHT 2,6-di-tert-butyl-4- methylphenol
  • the appropriate lipid standards were added based on the amount of DNA of the original sample (per mg DNA: 150 ⁇ PC 26:0; 50 ⁇ PC 28:0; 150 ⁇ PC 40:0; 75 ⁇ PE 28:0; 8,61 ⁇ PI 25:0 and 3nmol PS 28:0).
  • DNA 150 ⁇ PC 26:0; 50 ⁇ PC 28:0; 150 ⁇ PC 40:0; 75 ⁇ PE 28:0; 8,61 ⁇ PI 25:0 and 3nmol PS 28:0.
  • the lower organic fraction was collected using a glass Pasteur pipette and evaporated using a Savant Speedvac spdlllv (Thermo Fisher Scientific, Waltham, MA). The remaining lipid pellet was stored at -20°C.
  • lipid pellets were reconstituted in running solution (CH30H:CHCI3:NH40H; 90:10:1.25, v/v/v) depending on the amount of DNA of the original tissue sample ( ⁇ running solution/ ⁇ g DNA).
  • PL species were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a hybrid quadrupole linear ion trap mass spectrometer (4000 QTRAP system; Applied Biosystems, Foster City, CA) equipped with an Advion TriVersa robotic nanosource for automated sample injection. (Advion Biosciences, Ithaca, NY). Before measurement, samples were diluted in running solution.
  • PC phosphatidylcholine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PE phosphatidylethanolamine
  • PL profiles were recorded in positive and negative ion scan mode at a collision energy of 50eV for precursor (prec.) 184, 35eV for neutral loss (nl.) 141, -40eV for nl. 87 and -55eV for prec. 241 for PC, PE, PS and PI species respectively.
  • prec. precursor
  • nl. neutral loss
  • PI nl. 87 and -55eV for prec. 241 for PC, PE, PS and PI species respectively.
  • MRM mode Typically, a 3min period of signal averaging was used for each spectrum. Data were corrected for carbon isotope effects and are expressed as the percentage of total measurable PL species of the same PL family. Only the PL species which have an intensity of at least 5 times the blanc value were taken into account.
  • Clustering analysis was carried out by using an average linkage clustering algorithm of the Cluster 3.0 software [Eisen, M.B., P.T. Spellman, P.O. Brown, and D. Botstein, Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A, 1998. 95(25): p. 14863-8.].
  • the clustering results were visualized using the Java TreeView 1.1.5. software [Saldanha, A.J., Java Treeview-extensible visualization of microarray data.Bioinformatics, 2004. 20(17): p. 3246-8.].

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Abstract

L'invention concerne des procédés et des compositions pour le diagnostic et le traitement de cancers, par exemple, un cancer à cellules rénales, un cancer de la prostate, un cancer gastro-intestinal et un cancer colorectal. Les procédés et compositions sont associés aux phénotypes d'élongation de chaîne d'acide gras observés dans diverses tumeurs, telle que l'élongation de chaîne d'acide gras à médiation par les gènes de la voie d'élongation.
PCT/EP2013/056790 2012-03-28 2013-03-28 Enzymes d'élongation d'acide gras en tant que cibles pour le diagnostic et la thérapeutique du cancer WO2013144325A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110229905A (zh) * 2019-06-28 2019-09-13 徐州医科大学 Mcd基因及其表达产物在制备诊疗肾癌分期或转移试剂中的应用
CN110358763A (zh) * 2019-07-30 2019-10-22 云南大学 前列腺癌细胞LNCaP的FASN基因长短不同转录本的获取及定量方法
KR102156217B1 (ko) * 2019-03-05 2020-09-15 연세대학교 산학협력단 대장암 진단용 지질 바이오마커 및 이의 용도

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1552845A1 (fr) * 2002-06-26 2005-07-13 Takeda Pharmaceutical Company Limited Substances destinees a la prevention et/ou au traitement du cancer
US20070026424A1 (en) * 2005-04-15 2007-02-01 Powell Charles A Gene profiles correlating with histology and prognosis
US20070037204A1 (en) * 2003-08-08 2007-02-15 Hiroyuki ABURANTAI Gene overexpressed in cancer
US7989612B2 (en) 2002-02-20 2011-08-02 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7989612B2 (en) 2002-02-20 2011-08-02 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
EP1552845A1 (fr) * 2002-06-26 2005-07-13 Takeda Pharmaceutical Company Limited Substances destinees a la prevention et/ou au traitement du cancer
US20060189518A1 (en) 2002-06-26 2006-08-24 Takeda Pharmaceutical Company Limited Preventing/remedies for cancer
US20070037204A1 (en) * 2003-08-08 2007-02-15 Hiroyuki ABURANTAI Gene overexpressed in cancer
US20070026424A1 (en) * 2005-04-15 2007-02-01 Powell Charles A Gene profiles correlating with histology and prognosis

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
ABDEL-RAHMAN N. ZEKRI ET AL: "Molecular Prognostic Profile of Egyptian HCC Cases Infected with Hepatitis C Virus", ASIAN PACIFIC JOURNAL OF CANCER PREVENTION, vol. 13, no. 11, 30 November 2012 (2012-11-30), pages 5433 - 5438, XP055071434, ISSN: 1513-7368, DOI: 10.7314/APJCP.2012.13.11.5433 *
ALLSHIRE, SCIENCE, vol. 297, 2002, pages 1818 - 1819
BERSTEIN ET AL., NATURE, vol. 409, 2001, pages 363
EISEN, M.B.; P.T. SPELLMAN; P.O. BROWN; D. BOTSTEIN: "Cluster analysis and display of genome-wide expression patterns", PROC NATL ACAD SCI USA, vol. 95, no. 25, 1998, pages 14863 - 8, XP002939285, DOI: doi:10.1073/pnas.95.25.14863
ELBASHIR ET AL., EMBO J., vol. 20, 2001, pages 6877
FIRE ET AL., TRENDS GENET., vol. 15, 1999, pages 358
HALL ET AL., SCIENCE, vol. 297, 2002, pages 2232 - 2237
HILVO ET AL.: "Novel theranostic opportunities offered by characterization of altered membrane lipid metabolism in breast cancer progression", CANCER RES., vol. 71, no. 9, 2011, pages 3236 - 45, XP055071277, DOI: doi:10.1158/0008-5472.CAN-10-3894
HUTVAGNER ET AL., SCIENCE, vol. 293, 2001, pages 834
JENUWEIN, SCIENCE, vol. 297, 2002, pages 2215 - 2218
JUMP: "Mammalian Fatty Acid Elongases", METHODS MOL BIOL., vol. 579, 2009, pages 375 - 389, XP009171245, DOI: doi:10.1007/978-1-60761-322-0_19
KENJI TAMURA ET AL: "Novel Lipogenic Enzyme ELOVL7 Is Involved in Prostate Cancer Growth through Saturated Long-Chain Fatty Acid Metabolism", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 69, no. 20, 15 October 2009 (2009-10-15), pages 8133 - 8140, XP002634191, ISSN: 0008-5472, [retrieved on 20091013], DOI: 10.1158/0008-5472.CAN-09-0775 *
M. HILVO ET AL: "Novel Theranostic Opportunities Offered by Characterization of Altered Membrane Lipid Metabolism in Breast Cancer Progression", CANCER RESEARCH, vol. 71, no. 9, 1 May 2011 (2011-05-01), pages 3236 - 3245, XP055071277, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-10-3894 *
NYKANEN ET AL., CELL, vol. 107, 2001, pages 309
ROMANUIK ET AL.: "Novel biomarkers for prostate cancer including noncoding transcripts", AM J PATHOL., vol. 175, no. 6, 2009, pages 2264 - 2276, XP009171221, DOI: doi:10.2353/ajpath.2009.080868
ROMANUIK TAMMY L ET AL: "Novel biomarkers for prostate cancer including noncoding transcripts", AMERICAN JOURNAL OF PATHOLOGY, THE, AMERICAN SOCIETY FOR INVESTIGATIVE PATHOLOGY, vol. 175, 1 December 2009 (2009-12-01), pages 2264 - 2276, XP009171221, ISSN: 1525-2191, DOI: 10.2353/AJPATH.2009.080868 *
SALDANHA, A.J.: "Java Treeview--extensible visualization of microarray data", BIOINFORMATICS, vol. 20, no. 17, 2004, pages 3246 - 8
TAMURA ET AL.: "Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism", CANCER RES., vol. 69, no. 20, 2009, pages 8133 - 8140, XP002634191, DOI: doi:10.1158/0008-5472.CAN-09-0775
TANAKA ET AL., PREVENTING/REMEDIES FOR CANCER
USMAN ET AL., NUCLEIC ACIDS SYMP. SER., vol. 31, 1994, pages 163
USMAN; CEDERGREN, TIBS, vol. 17, 1992, pages 34
VOLPE ET AL., SCIENCE, vol. 297, 2002, pages 1833 - 1837

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102156217B1 (ko) * 2019-03-05 2020-09-15 연세대학교 산학협력단 대장암 진단용 지질 바이오마커 및 이의 용도
CN110229905A (zh) * 2019-06-28 2019-09-13 徐州医科大学 Mcd基因及其表达产物在制备诊疗肾癌分期或转移试剂中的应用
CN110229905B (zh) * 2019-06-28 2020-04-03 徐州医科大学 Mcd基因及其表达产物在制备诊疗肾癌分期或转移试剂中的应用
CN110358763A (zh) * 2019-07-30 2019-10-22 云南大学 前列腺癌细胞LNCaP的FASN基因长短不同转录本的获取及定量方法

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