EP1146903A1 - Glypicanes servant a detecter et a traiter le carcinome humain - Google Patents

Glypicanes servant a detecter et a traiter le carcinome humain

Info

Publication number
EP1146903A1
EP1146903A1 EP99954963A EP99954963A EP1146903A1 EP 1146903 A1 EP1146903 A1 EP 1146903A1 EP 99954963 A EP99954963 A EP 99954963A EP 99954963 A EP99954963 A EP 99954963A EP 1146903 A1 EP1146903 A1 EP 1146903A1
Authority
EP
European Patent Office
Prior art keywords
glypican
cells
growth factor
binding
expression
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.)
Withdrawn
Application number
EP99954963A
Other languages
German (de)
English (en)
Other versions
EP1146903A4 (fr
Inventor
Arthur Lander
Murray Korc
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.)
University of California
Original Assignee
University of California
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 University of California filed Critical University of California
Publication of EP1146903A1 publication Critical patent/EP1146903A1/fr
Publication of EP1146903A4 publication Critical patent/EP1146903A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04011—Phosphoinositide phospholipase C (3.1.4.11)
    • 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
    • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present application concerns medical sciences and more particularly detection and treatment of human cancers.
  • HSPGs heparan sulfate proteoglycans
  • GPI glycosylphophatidylinositol
  • HSPGs cell-surface heparan sulfate proteoglycans
  • HSPGs fibroblast growth factors
  • FGFs fibroblast growth factors
  • HB-EGF heparin-binding EGF-like growth factor
  • HGF hepatocyte growth factor
  • Wnt family of secreted glycoproteins 26, 88, 34, and 84.
  • VEGF vascular endothelial growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ s transforming growth factor- ⁇ s
  • BMPs bone morphogenetic proteins
  • Pancreatic cancer is responsible for over 20% of deaths due to gastrointestinal malignancies, making it the fourth to fifth most common cause of cancer related mortality.
  • the prognosis of patients with pancreatic cancer is extremely poor, with 5- year survival rates lower than 5% (105).
  • the reasons for this biological aggressiveness have not been clearly elucidated. Nonetheless, previous work has established that these cancers overexpress many mitogenic growth factors and their receptors (41) including a number of heparin-binding growth factors such as FGF1, FGF2, FGF5, HB-EGF, and amphiregulin. (108, 19, 4, and 44).
  • Breast cancer remains the second most common cause of cancer death in women in the United States (47). A variety of molecular alterations have been reported in breast cancer.
  • c-erbB-2 overexpression has been correlated with aggressive disease and decreased patient survival.
  • anti-erbB-2 antibodies can suppress breast cancer cell growth in vitro and decrease tumor burden in vivo, thereby prolonging patient survival (85, 40, 5, and 71).
  • c-erbB-2 is capable of heterodimerizing with the other members of the EGF receptor family once these receptors bind their ligands (92, 67, 108, and 104).
  • These ligands include epidermal growth factor (EGF), transforming gorwth factor-alpha (TGF-), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, amphiregulin (AR) and epiregulin.
  • EGF epidermal growth factor
  • TGF- transforming gorwth factor-alpha
  • HB-EGF heparin-binding EGF-like growth factor
  • AR amphiregulin
  • epiregulin heparin-binding factors.
  • breast cancers overexpress fibroblast growth factor-2 (FGF-2) and typel - 4 FGF receptors (10), and hepatocyte growth factor (HGF) and its receptor (c-Met) (be 19). Both FGF-2 and HGF are heparin-binding factors. Together, these observations suggest that multiple heparin-binding growth factors participate in the pathobiology of breast cancer in humans.
  • FGF-2 fibroblast growth factor-2
  • HGF hepatocyte growth factor
  • c-Met hepatocyte growth factor
  • glypican-1 expression - both mRNA and protein - is dramatically up-regulated in human cancers.
  • glypican-1 is highly expressed by human pancreatic carcinoma cell lines in vitro, and that in such cells glypicans (and no other classes of HSPGs) are uniquely required for FGF2 and
  • HSPG glypican-1 is strongly expressed in human breast and pancreatic cancer - both by the cancer cells and in the case of pancreatic cancer the adjacent fibroblasts - whereas expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis.
  • PI-PLC phosphoinositide-specific phospholipase-C
  • PI-PLC did not alter the response to the non-heparin binding growth factors EGF and insulin-like growth factor- 1 (IGF-1).
  • IGF-1 insulin-like growth factor- 1
  • HB-EGF heparin-binding epidermal growth factor-like growth factor
  • FGF-2 fibroblast growth factor-2
  • glypican-1 antisense construct attenuated glypican-1 protein levels and the mitogenic response to FGF2 and HB-EGF.
  • glypican-1 plays an essential role in the responses of pancreatic and breast cancer cells, and most likely in other glypican-responsive carcinomas as well, to certain mitogenic stimuli, that it is relatively unique in relation to other HSPGs.
  • Glypican can be used to detect the carcinoma in vitro and therapeutics that either bind to (e.g. , antibodies or drugs), remove (e.g. , enzymes) or prevent the expression (e.g., antisense constructs) of surface of the extracellular domain of glypican-1 are effective in retarding the growth of glypican-responsive carcinomas.
  • Figure 1 shows expression of glypican-1 in pancreatic tissues.
  • Total RNA (20 ⁇ g/lane) from 6 normal pancreatic tissues, and 8 pancreatic cancers (A), and from five normal and seven chronic pancreatitis tissues (B) were subjected to Northern blot analysis using a 32 P-labeled glypican-1 cDNA probe (500,000 cpm/ml).
  • a 7S ribosomal cDNA probe (50,000 cpm/ml) was used as a loading and transfer control. Exposure times were 2 days for glypican-1 and 6 h for 7S.
  • C glypican-1 protein expression in pancreatic cancers.
  • Membrane preparations (30 ⁇ g/lane) from 3 pancreatic cancer tissue samples were incubated in the absence (-) or presence (+) of heparitinase for 6 h at 37°C.
  • Western blotting was carried out with an affinity purified rabbit anti-rat glypican-1 antibody (250 ng/ml).
  • Figure 2 shows relative expression of glypican-1 mRNA. Autoradiographs of Northern blots for glypican-1 and 7S RNA from 12 normal (), 14 chronic pancreatitis
  • Figure 3 shows immunohistochemistry and in situ hybridization analysis of glypican-1 expression in human pancreatic cancer. Strong glypican-1 immunoreactivity was present in the fibroblasts surrounding the cancer cells (A,D), but not in the fibroblasts that were more distant from the tumor (A, arrowheads). Strong Glypican-1 mRNA in situ hybridization signals were present in both the cancer cells and in the fibroblasts immediately adjacent to the cancer cells (B,E). In contrast, glypican-1 mRNA expression was not evident in the fibroblasts distant from the tumor (B, arrowheads). In situ hybridization with a glypican-1 sense probe did not reveal any specific signal (C). Panel F provides a high power magnification, revealing faint glypican-1 immunoreactivity in the ductal like cancer cells. Scale bar: 50 ⁇ m (A-E), 2 ⁇ m (F).
  • Figure 4 shows expression of glypicans in cultured human pancreatic cancer cells and human placenta.
  • Northern blots of total RNA (20 ⁇ g/lane) isolated from the indicated cell lines and placenta (A-C) or polyA + RNA (2 ⁇ g/lane) isolated from fetal (D) and adult (E) brain tissue were hybridized with 32 P-labeled glypican-1, -2, -3, -4, -5 cDNA probes (500,000 cpm/ml) and with a 7S cDNA probe (50,000 cpm/ml).
  • A expression of glypican-1
  • B expression of glypican-3
  • C expression of glypican-4
  • D positive control for glypican-2 (human fetal brain tissue)
  • E positive control for glypican-5 (human adult brain tissue).
  • the 28S andl ⁇ S rRNA marker (A-C) and RNA size markers (D, E) are indicated by arrows.
  • Figure 5 shows glypican-1 protein expression in pancreatic cancer cell lines.
  • D 2.5 mg of total cell lysates prepared from PANC- 1 cells were subjected to anion exchange chromatography and incubated in the absence (-) or presence (+) of heparitinase.
  • E PANC-1 cells were incubated for 48 h in serum-free medium. The conditioned serum-free medium (100 ml) was subjected to anion exchange chromatography and the eluted fractions were incubated in the presence (+) or absence (-) of heparitinase. Immunoblot analysis was carried out with an affinity purified rabbit anti-rat glypican-1 antibody (250 ng/ml) that also recognizes human glypican-1 (A-E).
  • Figure 6 shows the effects of cleavage of GPI anchors on growth factor action in pancreatic cancer cells.
  • PANC-1 (A) and COLO-357 (B) cells were incubated with the indicated concentrations of EGF, HB-EGF, IGF-1, and FGF2 in the absence () o r presence (p) of PI-PLC as described in the Methods section. Data are expressed as percent increase or decrease of the respective untreated controls and are means ⁇ SEM of 8 determinations/experiment from three separate experiments.
  • Figure 7 shows: A: Expression of the modified glypican-1, glypl-VSVGTMR, in PANC-1 cells.
  • RNA isolated from wild-type PANC-1 cells, sham-transfected PANC-1 cells, and three pCDNA3.1/glypl-VSVGTMR clones (20 ⁇ g) were subjected to Northern blot analysis using a 32 P-labeled glypican-1 cDNA probe (500,000 cpm/ml). Exposure time was 12 h. Low levels of the endogenous glypican-1 transcript (3.5 kb) are seen in all cells. High levels of the glypl-VSVGTMR transcript (2.5 kb) are seen only in the transfected clones.
  • Sham transfected (A-D) and glypl-VSVGTMR transfected PANC-1 cells (E-H) were incubated for 1 h in the absence (A,B,E,F) or presence (C,D,G,H) of PI-PLC (0.5 U/ml), and stained with an affinity purified rabbit anti-rat glypican-1 antibody followed by addition of Cy3-conjugated anti-rabbit antibody.
  • Immunofluorescence (A,C,E,G) and the corresponding phase-contrast images (B,D,F,H) are shown. Scale bar: 30 ⁇ m.
  • Figure 9 shows effects of glypl-VSVGTMR on PI-PLC mediated inhibition of heparin-binding growth factor action.
  • Wild-type PANC-1 cells, sham-transfected PANC-1 cells, and three pCDNA3.1/glypl-VSVGTMR transfected PANC-1 clones were incubated with the indicated concentrations of HB-EGF (A), and FGF2 (B) in the absence (•) or presence ( ⁇ ) of PI-PLC as described in the Methods section.
  • Data are expressed as percent increase or decrease of unstimulated controls and are means ⁇ SEM of 8 determinations/experiment from three separate experiments.
  • Figure 10 shows expression of glypican-1 antisense (Gl-AS-1751) mRNA and glypican-1 protein in PANC-1 cells.
  • Total RNA (20 ⁇ g/lane) and total cell lysates (30 ⁇ g/lane) were isolated from PANC-1 cells at the indicated times after transfection with the indicated amounts of pMH6/Gl-AS-1751 plasmid DNA (the total amount of transfected DNA was equal in all samples).
  • A: Northern blots were hybridized with a 32 P-labeled glypican-1 sense riboprobe (500,000 cpm/ml). Exposure time was 6 h. Equal loading was determined by ethidium bromide staining.
  • PANC-1 cells (•), PANC-1 cells transfected with equivalent amounts of pMH6 DNA (A), and Gl-AS-1751 transfected PANC-1 cells (D) were incubated for 48 h with the indicated concentrations of EGF, HB-EGF, IGF-1, and FGF2. Data are expressed as percent change from unstimulated controls and are means ⁇ SEM of 8 determinations/experiment from three separate experiments.
  • Figure 12 shows : A: Glypican-1 protein expression in PANC-1 pancreatic cancer cells. 30 ⁇ g of total cell lysates were incubated in the absence (-) or presence (+) of heparitinase. Immunoblot analysis was carried out with an affinity purified rabbit anti-rat glypican-1 antibody (250 ng/ml) that also recognizes human glypican-1. Visualization was performed by enhanced chemiluminescence (ECL).
  • Middle and lower panel Expression of glypican-1 mRNA in the indicated PANC-1 clones.
  • Northern blots of total RNA (20 ⁇ g/lane) isolated from the indicated cell lines were hybridized with a 32 P-labeled glypican-1 antisense riboprobe (middle panel), and with a glypican-1 sense riboprobe (lower panel). Equal loading was verified by ethidium bromide staining of the gel.
  • Figure 13 shows the effects of decreased endogenous glypican-1 levels on heparin-binding growth factor responsiveness in PANC-1 cells.
  • Parental (•), sham transfected ( ⁇ ), and 4 glypican-1 antisense mRNA expressing PANC-1 clones (open symbols) were incubated for 48 h with the indicated concentrations of HB-EGF, FGF2, and HGF (in nM).
  • Data are expressed as percent change from unstimulated controls and are means ⁇ SEM of 8 determinations per experiment from three separate experiments.
  • Figure 14 shows the effects of decreased endogenous glypican-1 levels on non- heparin-binding growth factor responsiveness in PANC-1 cells.
  • Figure 15 shows the effects of decreased endogenous glypican-1 levels on in vivo tumorigenicity in PANC-1 cells.
  • Exponentially growing parental, sham, and glypican-1 antisense transfected PANC-1 cells (lxlO 6 ) were injected subcutaneously in athymic nude mice and tumor growth was measured weekly until week 8.
  • A: Tumor volume was determined by the equation: vol (1 x h x w) x ⁇ /4, where 1 is length, h is height, and w is width of the tumor.
  • Figure 16 shows expression of glypicans and syndecan-1 in breast tissues.
  • Total RNA (20 ⁇ g/lane) from four normal breast tissues (N) and eight breast cancers (Ca) were subjected to Northern blot analysis using a 32 P-labeled glypican-1 (A), -3 (B), -4 (C) and syndecan-1 (D) cDNA probes (500000 cpm/ml).
  • a 7S ribosomal cDNA probe (50000 cpm/ml) was used as a loading and transfer control. Exposure times were 1 day for glypican-1, -3 and syndecan-1, 2 days for glypican-4 and 6 hours for 7S. Arrows indicate the two glypican-4 mRNA transcripts (3.4 and 4.6 kb).
  • Figure 17 shows relative expression of glypican-1, glypican-3 and syndecan-1 mRNA.
  • Autoradiographs of Northern blots for glypican-1, glypican-3 or syndecan-1 and 7S RNA from 10 normal and 20 cancerous breast tissue samples were analyzed by densitometry and the level of glypican-1, glypican-3 or syndecan-1 expression was calculated as the ratio of glypican-1 ( ⁇ ), glypican-3 (A) or syndecan-1 ( ⁇ ) and 7S RNA. Closed symbols are high expressing groups of breast cancer tissues for glypican- 1, glypican-3 and syndecan-1.
  • Figure 18 shows expression of glypican-1 in human breast cancer tissue.
  • A Immunostaining revealed strong glypican-1 immunoreactivity in the intraductal carcinomas.
  • D In the lobular part, faint glypican-1 immunoreactivity was present in these cancer cells.
  • B, E In situ hybridization analysis of serial sections revealed strong glypican-1 mRNA signals in the lobualr part.
  • C, F Hybridization with the glypican-1 sense probe did not yield any specific signals. Magnification, 200.
  • Figure 19 shows mild hyperplasia of usual type with heaped-up cells which have "snout” like luminal aspect of cells. Note at upper right an artifact of histological sectioning which produced apparent filling of a portion of the space. Immunostaining revealed only faint glypican-1 immunoreactivity in these ductal cells (A), moderate glypincan-1 immunoreactivity in the fibroblast or myoepithelial cells surrounding the lumen (A inset). In situ hybridization analysis of serial sections revealed strong glypican-1 mRNA signals in the ductal cells (B). Hybridization with the sense glypican-1 prove did not yield any specific signals (C). Magnification 200.
  • Figure 20 shows glypican-1 expression in breast cancer cell lines.
  • A Northern blots of total RNA (20 ⁇ g/lane) isolated from MDA-MB-231 and MDA-MB-468 cells were hybridized with 32 P-labeled glypican-1 cDNA probe (500000 cpm ml) and with a 7S cDNA probe (50000 cpm/ml). Exposure times were 1 day for glypican-1 and 6 hours for 7S.
  • 30 ⁇ g of total lysates (B) and 30 ⁇ g of membrane proteins (C) prepared from MDA-MB-231 and MDA-MB-468 cells were incubated in the absence (-) or presence (+) of heparitinase.
  • MDA-MB-231 and MDA-MB-468 cells were incubated for 48 hours in serum-free medium.
  • the conditioned serum-free medium 100 ml
  • the conditioned serum-free medium 100 ml
  • the eluted fractions were incubated in the absence (-) and presence (+) of heparitinase.
  • Immunoblot analysis was carried out with an affinity purified rabbit anti-rat glypican-1 antibody (250 ng/ml) that also recognizes human glypican-1 (B-D).
  • Figure 21 shows 30 ⁇ g of total cell lysates prepared from MDA-MB-231 and MDA-MB-468 cells were subjected to heparitinase digestion and analyzed by SDS- PAGE under nonreducing and reducing conditions as described in Methods.
  • Figure 22 shows syndecan-1 expression in breast cancer cell lines.
  • A Northern blots of total RNA (20 ⁇ g/lane) isolated from MDA-MB-231 and MDA-MB- 468 cells were hybridized with 32 P-labeled syndecan-1 cDNA probe (500000 cpm/ml) and with a 7S cDNA probe (50000 cpm ml). Exposure times were 1 day for syndecan- 1 and 6 hours for 7S.
  • B 30 ⁇ g of total lysates were prepared from MDA-MB-231 and MDA-MB-468 cells.
  • C MDA-MB-231 and MDA-MB-468 cells were incubated for 48 hours in serum-free medium. The conditioned serum-free medium (100 ml) were subjected to anion exchange chromatography. Immunoblot analysis was carried out with human anti-mouse syndecan-1 antibody.
  • Figure 23 shows the effects of cleavage of GPI anchors on growth factor action in breast cancer cells.
  • MDA-MB-231 and MDA-MB-468 cells were incubated with the indicated concentrations of IGF-1, HB-EGF and FGF-2 in the absence or presence of PI-PLC as described in Methods. Data are expressed as percent increase or decrease of the respective untreated controls and are meansSEM of eight determinations per experiment from three separate experiments (#: p ⁇ 0.001, *: p ⁇ 0.05). **: p ⁇ 0.05,
  • Figure 24 shows expression of glypican-1 mRNA in the indicated MDA-MB-
  • Figure 25 shows the effects of decreased endogenous glypican-1 levels on growth factor responsiveness in MDA-MB-231 cells.
  • Parental (•), sham transfected (A), and 4 glypican-1 antisense (GAS) mRNA expressing MDA-MB-231 clones (open symbols) were incubated for 48 hours with the indicated concentrations of EGF, IGF- 1, HB-EGF, FGF-2 and HGF.
  • Data are expressed as percent change from unstimulated controls and are means SEM of 8 determinations per experiment from three separate experiments (*: p ⁇ 0.05).
  • Figure 26 shows the effects of decreased endogenous glypican-1 levels on growth factor responsiveness in MDA-MB-468 cells.
  • Parental (•) and 1 glypican-1 antisense (GAS) mRNA expressing MDA-MB-468 clone (O) were incubated for 48 hours with the indicated concentrations of EGF, IGF-1, HB-EGF, FGF-2 and HGF.
  • Data are expressed as percent change from unstimulated controls and are means SEM of 8 determinations per experiment from three separate experiments ($: p ⁇ 0.005, #: p ⁇ 0.01, *: p ⁇ 0.05).
  • FBS FBS, DMEM and RPMI medium, trypsin solution, penicillin-streptomycin solution, and Geneticin (G418) from Irvine Scientific (Santa Ana, CA); Genescreen membranes from New England Nuclear (Boston, MA); restriction enzymes, pMH6 vector, the random primed labeling kit, the Genius 3 non-radioactive nucleic acid detection kit, and the Genius 4 RNA labeling kit from Boehringer-Mannheim (Indianapolis, IN); phosphoinositide-specific phospholipase-C (PI-PLC) form Oxford Glycosciences Inc.
  • pancreatic tissue samples (7 male, 5 female donors; median age 41.8 years; range 14-68 years), chronic pancreatitis tissues (13 male, 1 female; median age 42.1 years; range 30-56 years), and pancreatic cancer tissues (10 male, 6 female; median age 62.6 years; range 53-83 years) were obtained through an organ donor program and from surgical specimens obtained from patients with severe symptomatic chronic pancreatitis or from pancreatic cancer patients.
  • pancreatic cancer tissues 10 male, 6 female; median age 62.6 years; range 53-83 years
  • TNM classification of the Union Internationale Contre le Cancer (UICC) 6 tumors were stage 1, 1 stage 2, and 9 stage 3 ductal cell adenocarcinoma.
  • Freshly removed tissue samples were fixed in 10% formaldehyde solution for 12-24 h and paraffin- embedded for histological analysis.
  • tissue samples were frozen in liquid nitrogen immediately upon surgical removal and maintained in -80°C until use for RNA extraction. All studies were approved by the Ethics Committee of the University of Bern and by the Human Subject
  • Breast cancer tissues (20 female patients; median age: 60.8 yr; range: 38-76 yr) were obtained from surgical specimens from patients with breast cancer.
  • Normal human breast tissue samples (10 female patients; median age: 63.2 yr ; range: 38-76 yr) were obtained from the same mastectomy samples, but at a distance of at least 5 cm from the cancer area.
  • 10 tumors were stage 1, 7 tumors were stage 2 and 3 tumors were stage 3 breast carcinoma.
  • Freshly removed tissue samples were fixed in 10% formaldehyde solution for 12-24 h and paraffin embedded for histological analysis.
  • tissue samples were frozen in liquid nitrogen immediately upon surgical removal and maintained in -80 until used for RNA extraction. All studies were approved by the Ethics Committee of the Yamanashi-Medical University and by the Human Subjects Committee at the University of California, Irvine.
  • a 599 bp human glypican cDNA probe (nucleotides 920-1518) was isolated as described previously (59) and subcloned into Bluescript-HSK-r- vector.
  • a 210 bp cDNA fragment (nucleotides 1280-1489) of human glypican was subcloned into Bluescript-IISK+ vector. Authenticity was confirmed by sequencing.
  • Glypican-2 and glypican-5 constructs were prepared as described previously (100, 91).
  • a 239 bp human glypican-3 cDNA fragment, corresponding to nt 927 to 1165 of the human glypican-3 cDNA sequence (Genbank: HSU50410), and a 273 bp human glypican-4 cDNA fragment, corresponding to nt 12 to 284 of a human glypican-4 EST sequence (Genbank: AA046130) were generated by RT-PCR from human placenta RNA.
  • a glypican-1 antisense construct was prepared by RT-PCR amplification of human placenta cDNA as described previously (84).
  • the 1751 bp fragment (nt 123-1873; Genbank accession X54232), that covered from 100 bp downstream of the start codon to 25 bp downstream of the end of the coding region, was subcloned in the antisense orientation into the pMH expression vector.
  • the primers used for the glypican-3/-4 preparation contained a EcoRI and BamHI site, respectively, attached to the '-end and preceded by a 3 bp overhang.
  • Glypican-3 sense 5'-AGT-GG rCC-CTGCTCTTACTGCCAGGGAC
  • antisense 5'-GTA- GA47TC-GCTTTCCTGCATTCTTCTGG.
  • Glypican-4 sense 5'-AGT-GG4rCC- GTTGACACCAGCAAACCAGA
  • antisense 5'-GTA-GA 7TC- AGTGAGGAGGTAGGCCTGTG.
  • Authenticity was confirmed by sequencing.
  • An eukaryotic expression vector that directs expression of a transmembrane version of glypican-1 was constructed by fusing the membrane domain of the vesicular stomatitis virus glycoprotein (VSVG) (1) with the extracellular domain of glypican-1.
  • VSVG vesicular stomatitis virus glycoprotein
  • VSVGTMR 80 bp fragment encoding the transmembrane domain of VSVG
  • VSVGTMR 80 bp fragment encoding the transmembrane domain of VSVG
  • a BamHI- Pmll 1.7 kb fragment, corresponding to the extracellular domain of the rat glypican-1 was inserted into the pCDNA3.1-myc-His [VSVGTMR] expression vector by directional cloning.
  • the construct was termed glypl-VSVGTMR.
  • Authenticity was confirmed by sequencing.
  • the final result contained aminoacids 1-539 of rat glypican- 1 followed by HVSIASFFFIIGLIIGLFVVLKLSRGPFE QKLISEEDLNMHTGHHHHHH.
  • a glypican-1 antisense construct was prepared by PCR amplification of human placenta cDNA.
  • the 1751 bp fragment (nt 123-1873), which covered from 100 bp downstream of the start codon to 25 bp downstream of the end of the coding region, was subcloned in the antisense orientation into the pMH6 expression vector.
  • the primers used for the glypican-1 preparation contained a EcoRI and Hindlll site, respectively, attached to the 5'-end and preceded by a 3 bp overhang.
  • Sense 5'-GTA-GA47TC-GGACCTTGGCTCTGCCCTTC
  • antisense 5'-AGT- 4GC7T-GTAAGGGCCAGGAAGAGGAG.
  • the construct was termed Gl-AS-1751. Authenticity was confirmed by sequencing.
  • RNA extraction and Northern blot analysis Total RNA was extracted by the single step acid guanidinium thiocyanate phenol chloroform method. RNA was size fractionated on 1.2% agarose/ 1.8 M formaldehyde gels, electrotransferred onto nylon membranes and cross-linked by UV irradiation. Blots were pre-hybridized and hybridized with cDNA probes or riboprobes and washed under high stringency conditions as previously reported (42). Blots were then exposed at -80°C to Kodak XAR-5 films and the resulting autoradiographs were scanned to quantify the intensity of the radiographic bands. A BamHI 190 bp fragment of mouse 7S cDNA that hybridizes with human cytoplasmatic RNA was used to confirm equal RNA loading and transfer (42).
  • pancreatic cancer or breast cancer chronic pancreatitis and normal pancreatic tissues were subjected to immunostaining using the streptavidin-peroxidase technique. Endogenous peroxidase activity was blocked by incubation for 30 min with
  • Immunoblotting Cells were washed with PBS (4°C) and solubilized in lysis buffer containing 50 mM Tris-Hcl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 ⁇ g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 % Triton X-100. Digestion with heparitinase (1 U/ml) was performed in a volume of 30 ⁇ l at 37°C for 6 h and terminated by the addition of 7.5 ⁇ l 5x SDS sample buffer and heating at 95°C for 10 min.
  • cells or tissue samples were homogenized in 20 mM Hepes pH 7.4, 1.5 mM MgCl 2 , 1 mM EGTA, 1 mM PMSF, 2 mM benzamidine and centrifuged at 1500g for 10 min. Supernatants were collected and centrifuged at 25,000g for 30 min. Pellets were resuspended in 20 mM Hepes pH 7.4 containing 10 mM leupeptin. For reduction and alkylation with iodoacetamide, protein lysates were incubated at 95°C for 4 min in the presence of 10 mM DTT.
  • iodoacetamide was added to the samples to a final concentration of 50 mM and incubated at 95°C for 2 min. Proteins were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon P membranes. Membranes were incubated for 90 minutes with a polyclonal rabbit anti-rat glypican-1 (250 ng/ml) antibody, washed and incubated with a secondary antibody against rabbit IgG for 60 min. After washing, visualization was performed by enhanced chemiluminescence.
  • Glycanated glypican-1 was purified by anion exchange chromatography on DEAE-Sephacel equilibrated in buffer A (50 mM Tris-Hcl, pH 8.0, 0.15 M NaCl, 0.1 % Triton X-100).
  • Cell lysates in buffer B 50 mM Tris-Hcl, pH 8.0, 0.15 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 ⁇ g/ml pepstatin A, 1 mM PMSF
  • tissue sections (4 ⁇ m thick) were placed on 3-aminopropyl-methoxysilane-coated slides, deparaffinized and incubated at room temperature for 20 min with 0.2N HCl and for 15 min with 20 ⁇ g/ml proteinase K at 37 °C.
  • the sections were then post-fixed for 5 min in phosphate buffered saline (PBS) containing 4% paraformaldehyde, incubated briefly twice with PBS containing 2 mg/ml glycine and once in 50% (v/v) formamide/2x SSC for 1 h prior to initiation of the hybridization reaction by the addition of 100 ⁇ l of hybridization buffer.
  • PBS phosphate buffered saline
  • the hybridization buffer contained 0.6 M NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7.5), 0.25% SDS, 200 ⁇ g/ml yeast tRNA, lx Denhart's solution, 10% dextran sulfate, 40% formamide and 100 ng/ml of the indicated digoxigenin-labeled riboprobe.
  • Hybridization was performed in a moist chamber for 16 h at 42°C. The sections were then washed sequentially with 50% formamide/2x SSC for 30 min at 42°C, 2x SSC for 20 min at 42°C, and 0.2x SSC for 20 min at 42°C.
  • the Genius 3 nonradioactive nucleic acid detection kit was used for immunological detection. The sections were washed briefly with buffer 1 solution (100 mM Tris-HCl and 150 mM NaCl, pH 7.5) and incubated with 1 % (w/v) blocking reagents in buffer 1 solution for 60 min at room temperature.
  • the sections were then incubated for 30 min at room temperature with a 1:2000 dilution of alkaline phosphatase conjugated polyclonal sheep anti-digoxigenin Fab fragment antibody, washed twice for 15 min at room temperature with buffer 1 solution containing 0.2% Tween 20 and equilibrated for 2 min with buffer 2 solution (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl 2 , pH 9.5). The sections were then incubated with color solution containing nitroblue tetrazolium and X-phosphate in a dark box for 2-3 h. After the reaction was stopped with TE buffer (10 mM Tris-Hcl, pH 7.4, 1 mM EDTA, pH 8.0), the sections were mounted in aqueous mounting medium.
  • TE buffer 10 mM Tris-Hcl, pH 7.4, 1 mM EDTA, pH 8.0
  • FBS fetal bovine serum
  • penicillin 100 U/ml
  • streptomycin 100 ⁇ g/ml streptomycin (complete medium).
  • COLO-357 and PANC-1 cells were plated overnight at a density of 10,000 cells/well in 96-well plates, washed in Hank's buffered saline solution (HBSS), and subsequently incubated in serum-free medium (DMEM containing 0.1% BSA, 5 ⁇ g/ml transferrin, 5 ng/ml sodium selenite, and antibiotics) in the absence or presence of various growth factors.
  • HBSS Hank's buffered saline solution
  • DMEM serum-free medium
  • MTT 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
  • Human breast cancer cells were routinely grown in Leibovitz's Medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (complete medium).
  • MDA-MB-231 and MDA-MB-468 were plated overnight at a density of 10000 cells/well in 96-well plates, washed in HBSS, and subsequently incubated in serum-free medium (Leibovitz's Medium containing 0.1% BSA, 5 ⁇ g/ml transferrin, 5 ng/ml sodium selenite and antibodies) in the absence or presence of various growth factors.
  • serum-free medium Leibovitz's Medium containing 0.1% BSA, 5 ⁇ g/ml transferrin, 5 ng/ml sodium selenite and antibodies
  • PI-PLC cells were incubated with the indicated concentrations of PI-PLC for 1 hour.
  • Transient transfection of the glypican-1 antisense construct pMH6/Gl-AS-1750 and stable transfection of Gl-AS-1751 were carried out by the lipofectamine method.
  • Immunofluorescence Cells were plated at a density of 100,000 cells/slide on culture chamber slides and incubated for 24 h in complete medium. Subsequently, cells were incubated for 1 h in serum free medium in the absence or presence of PI-PLC (0.5 U/ml), washed, and incubated for 30 min at room temperature in DMEM containing 5% goat serum, 5% fetal bovine serum, and 10 mM Hepes pH 7.5. All antibody dilutions and washings were carried out at 4°C using this solution.
  • Glypican-1 expression in human pancreatic tissue Northern blot analysis was performed on total RNA isolated from 12 normal pancreatic tissues, 16 pancreatic cancers, and 14 chronic pancreatitis samples.
  • the 3.7 kb glypican-1 mRNA transcript was of relatively low abundance but clearly visible in 4 of 12 normal pancreatic tissue samples (Fig. 1A) and in 3 of 14 chronic pancreatitis samples. It was also faintly visible on the original autoradiographs in 7 normal and 9 chronic pancreatitis samples (Fig. IB).
  • 12 of 16 pancreatic cancer samples exhibited moderate to high levels of glypican-1 mRNA (Fig. 1A).
  • HSPGs including glypican-1
  • glypican-1 generally appear on immunoblots as broad high-molecular- weight smears having faint immunostaining, in part due to poor binding of proteoglycans to blotting membranes (35, 60, 81).
  • HSPGs migrate as distinct bands on SDS-PAGE.
  • membrane preparations from normal and cancerous pancreatic tissue (30 ⁇ g) were incubated in the absence or presence of heparitinase for 6 h at 37 °C and subjected to SDS-PAGE followed by Western blot analysis.
  • a single 55 kDa band corresponding to the glypican-1 core protein was seen in 4 of 6 pancreatic cancer samples following heparitinase digestion, but not in untreated samples.
  • membrane preparations from 4 normal pancreatic tissues did not exhibit a glypican-1 band even following heparitinase treatment.
  • An example of an immunoblot experiment with pancreatic cancer tissues is shown in Fig. IC.
  • RNA isolated from 10 normal breast tissues and 20 breast cancer samples The 3.7-kb glypican-1 mRNA transcript was of relatively low abundance, but clearly visible in 6 of 10 normal breast tissue samples. In contrast, 10 of 20 breast cancer samples exhibited moderate to high levels of glypican-1 mRNA. The glypican-3 mRNA transcript was expressed at moderate to high levels in 5 of 10 normal breast tissue samples. In the breast cancers, it was expressed at moderate to high levels in 6 of 20 samples. The glypican-4 mRNA transcripts were below the level of detection in the normal samples, and barely detectable in 7 of 20 breast cancer samples. A representative Northern blot is shown in Fig. 16 (A-C).
  • Glypican-2 and -5 mRNA transcripts were below the level of detection in both normal and cancer samples.
  • the same cDNAs used in present study are able to detect the presence of the corresponding glypican-2 and -5 mRNA transcripts in human brain RNA (37), indicating that the failure to detect these transcripts in breast tissues was not due to technical difficulties with these cDNAs.
  • syndecan-1 was also expressed at high levels in breast cancer.
  • the 3.4- and 2.6-kb syndecan-1 mRNA transcripts were present at low levels in all 10 normal breast tissue samples. However, with the exception of one sample, both transcripts were only clearly visible on the original autoradiographs. In contrast, in the breast cancers, both syndecan-1 transcripts were expressed at moderate to high levels in 9 of 20 samples (Fig. ID).
  • glypican-1 immunoreactivity was not present in the connective tissue cells or in the terminal ductal-lobular unit (data not shown).
  • Northern blot analysis revealed high levels of glypican-1 mRNA in T3M4 and COLO-357 cells, moderate to high levels in ASPC-1 cells, and moderate to low levels in human placenta and in CAPAN-1, MIA-PaCa-2, and PANC-1 pancreatic cancer cells (Fig. 4A).
  • the glypican-4 mRNA transcripts were barely detectable in the cancer cell lines, whereas glypican-2, -3, and -5 were below level of detection by Northern blot analysis in all the cancer cell lines (Fig. 4B, C, and data not shown).
  • Glypican-3 and - 4 were expressed at moderate to high levels in human placenta (Fig. 4B, C), whereas glypican-2, and -5 were also below level of detection in placenta RNA (data not shown).
  • glypican-2/5 signals in both the pancreatic cancer cell lines and in human placenta, hybridization of human multiple tissue Northern blots was carried out with the glypican-2/5 specific probes.
  • the glypican-2 transcript (approximately 4.0 kb, Fig. 4D) was evident in human fetal brain tissue (7) and the glypican-5 transcript (approximately 3.7 kb, Fig. 4E) was readily apparent in human adult brain tissue.
  • the 55 kDa band was observed in the total protein lysate sample as well as in the membrane preparation sample (Fig. 5B).
  • the 55 kDa band that was observed under reducing conditions migrated as a band of approximately 48 kDa under non-reducing conditions (Fig. 5C). This degree of shift is characteristic of the core proteins of the glypican family, which have many disulfide bonds and migrate more rapidly under non-reducing conditions then under reducing conditions (51).
  • conditioned serum-free medium from PANC-1 and COLO-357 cells was collected during a 48 h incubation, and subjected to anion exchange chromatography to isolate HSPGs. The eluted fractions were incubated in the presence or absence of heparitinase. Immunoblot analysis revealed the presence of the 55 kDa band, representing the glypican-1 core protein, in the heparitinase digested sample of PANC-1 (Fig. 5E) and COLO-357 cells (data not shown), indicating that both cells release glycanated glypican-1 into the culture medium.
  • Northern blot analysis revealed high levels of glypican-1 mRNA in both cell lines (MDA-MB-231 and MDA-MB-468)
  • glypican-2, -3, -4 and -5 were below the level of detection by Northern blot analysis in both cell lines.
  • Immunoblotting with a highly specific anti- glypican-1 antibody revealed the presence of a 55-kDa band corresponding to glypican-
  • MDA-MB-231 and MDA-MB-468 cells was collected during a 48-h incubation, and subjected to anion exchange chromatography to isolate HSPGs. Immunoblotting revealed the presence of the 55-kDa band, representing the glypican-1 core protein
  • membrane samples (30 ⁇ g) were subjected to heparitinase digestion and analyzed by SDS-PAGE under reducing and nonreducing conditions.
  • the 55-kDa band that was observed under reducing conditions migrated as a band of 48 kDa under nonreducing conditions (Fig. 21). This shift is characteristic of the core proteins of glypicans, since these proteins have many disulfide bonds and migrate more rapidly under nonreducing conditions than under reducing conditions (51).
  • COLO-357 and PANC-1 cells were incubated in the presence or absence of phosphoinositide-specific phospholipase-C (PI-PLC), which cleaves glypican-1 and other proteins that associate with membranes via a covalent GPI lipid linkage.
  • PI-PLC phosphoinositide-specific phospholipase-C
  • HB-EGF exerted a dose-dependent increase in cell proliferation.
  • Preincubation of either cell line with PI-PLC (0.5 U/ml) and subsequent incubation with the same concentrations of FGF2 or HB-EGF in the presence of PI-PLC (0.1 U/ml) completely blocked the stimulatory effect of these heparin-binding growth factors (Fig. 6A,B).
  • PI-PLC had no significant effect on the growth stimulatory actions of EGF and IGF-I, which are non-heparin binding growth factors (Fig. 6A,B).
  • PI-PLC In addition to removing glypican-1, PI-PLC removes other GPI-anchored proteins from the cell membrane. Therefore, we next sought to determine whether restoring glypican-1 expression could block the loss of responsiveness to heparin- binding growth factors that occurs following PI-PLC treatment.
  • PANC-1 cells were stable transfected with a glypican-1 construct that encodes the extracellular domain of glypican-1 fused to the transmembrane domain of the vesicular stomatitis virus G glycoprotein (VSVG), and which is therefore resistant to the actions of PI- PLC. This construct was also tagged at its C-terminus with a c-r ⁇ yc epitope.
  • Clones transfected with the pCDNA3.1/glypl-VSVGTMR construct exhibited a 2.5 kb transcript by Northern blot analysis (Fig. 7A). Expression of the fusion protein was confirmed by immunoblotting with an anti-c-myc antibody (Fig. 7B).
  • immunofluorescence was carried out in untreated control and pCDNA3.1 /glypl- VSVGTMR transfected PANC-1 cells and in the respective cells following PI-PLC treatment. Relatively low intensity signals corresponding to endogenous glypican-1 were present in parental PANC-1 cells, and these signals were further decreased by PI- PLC treatment (Fig. 8A-D). In contrast, glypl-VSVGTMR transfected PANC-1 cells exhibited strong glypican-1 immunofluorescence signals, which were not attenuated by PI-PLC treatment (Fig. 8E-G).
  • control and three clones expressing the glypl-VSVGTMR construct were next incubated with growth factors in the presence or absence of PI-PLC.
  • FGF2 and HB-EGF enhanced proliferation in a dose- dependent manner, and this effect was completely blocked by PI-PLC treatment (Fig. 9).
  • PI-PLC did not significantly alter the growth stimulatory actions of FGF2 and HB-EGF (Fig. 9).
  • glypl-VSVGTMR construct prevented PI-PLC from blocking heparin-binding growth factor responsiveness, the effects of PI-PLC in parental PANC-1 cells were most likely due to its ability to cleave endogenous glypican-1.
  • glypican-1 regulates growth factor action in breast cancer cells.
  • MDA-MB-231 and MDA-MB-468 cells were incubated in the absence or presence of PI-PLC.
  • IGF-1, HB-EGF and FGF-2 induced cell proliferation.
  • Preincubation of either cell line with PI-PLC (0.5 U/ml) and subsequent incubation with the same concentrations of each growth factor in the presence of PI-PLC (0.1 U/ml) completely blocked the stimulatory effect of HB-EGF and FGF-2.
  • PI-PLC had no significant effect on the growth stimulatory actions of IGF-1, which is not a heparin-binding growth factor (Fig. 23).
  • PI-PLC can remove many GPI-anchored proteins from the cell surface, we next sought to determine whether it is possible to modulate responsiveness to heparin-binding growth factors by altering endogenous glypican-1 protein levels.
  • Glypican-1 generally appears on immunoblots as a faint high-molecular- weight smear, due to the high heparan-sulfate content and subsequent poor binding to blotting membranes (35, 60, and 81).
  • HSPGs including glypican-1 migrate as distinct bands. Therefore, PANC-1 cell lysates were incubated in the absence or presence of heparitinase for 6 h at 37 °C and subjected to SDS-PAGE. A 55 kDa band corresponding to the glypican-1 core protein was visible in the absence or presence of heparitinase treatment (Fig. 12A).
  • heparin binding growth factors HB-EGF, FGF2, and HGF
  • 2 non-heparin binding growth factors EGF and IGF-1
  • FGF2, HB-EGF, and HGF enhanced proliferation in a dose-dependent manner in parental and sham transfected PANC-1 cells, and this stimulatory effect was markedly attenuated in the glypican-1 antisense transfected PANC-1 clones (Fig. 13).
  • the growth stimulatory actions of EGF and IGF-1 were similar in parental, sham transfected, and glypican-1 antisense transfected PANC-1 cells (Fig. 14).
  • HSPGs are thought to play an important role in growth factor signaling, a role that has been particularly well documented for FGFs (82, 109, and 7). HSPGs are essential for the interactions of FGFs with their high affinity receptors in a number of cell types, including CHO cells, 3T3 fibroblasts, lymphoid cells, myeloblasts (68), chondrocytes, and MCF-7 breast cancer cells (16). HSPGs may act by increasing the affinity of FGFs for their receptors, facilitating receptor dimerization and subsequent signaling, and/or stabilizing FGFs by protecting them from proteolysis or thermal denaturation. HSPGs have also been shown to be essential for mitogenic signaling of
  • HSPGs of the " glypican family - glypican-3 and Drosophila Dally - have been implicated in the control of cellular growth. Mutations in Dally produce morphological defects in certain fly tissues by affecting patterned cell divisions, and glypican-3 mutations cause the Simpson-Golabi-
  • HSPGs could be responsible for differences in their proliferative and invasive properties (16, 49).
  • HSPGs are known to interact with FGF-2, KGF, VEGF, HB-EGF and HGF.
  • HGF and FGF are known to regulate the morphogenesis and differentiation of mammary epitherial cells, and HSPGs are likely to be important regulators of the development of the gland.
  • HSPGs are differently distributed in normal and malignant breast epitherial cells, and this difference in HSPG distribution correlates with differences in sensitivity to FGF2.
  • the 55 kDa glypican-1 core protein was evident only following heparitinase treatment, which is in agreement with the observation in other tissues that intact glypican-1 migrates as a broad high-molecular-weight smear on SDS-PAGE.
  • the proliferative lesions exhibiting a pattern of intraductal epithelial hyperplasia did not harbor glypican-1 immunoreactivity.
  • the fibroblasts and myoepithelial cells immediately adjacent to the proliferative ductal cells exhibited moderate glypican-1 immunoreactivity.
  • in situ hybridization analysis revealed that strong glypican-1 mRNA signal was found in the proliferative ducts associated with intraductal epithelial hyperplasia. It is likely, therefore, that these proliferative lesions, like the breast cancer cells produce and release glypican-1.
  • heparin binding growth factors such as FGF2 (24) and HB-EGF.
  • FGF2 heparin binding growth factors
  • HB-EGF heparin binding growth factors
  • glypican-1 present on fibroblasts adjacent to the tumor may also act to dampen the mitogenic response to these growth factors.
  • glypican-1 enhances the mitogenic response to FGF1, while inhibiting the mitogenic response to FGF7.
  • inhibiting proteoglycan sulfation in MDA-MB-231 human breast cancer cells decreases binding of FGF2 to HSPGs and restores responsiveness to FGF2 mitogenic signals.
  • glypican-1 plays an important role in FGF2 and HB-EGF signaling in pancreatic carcinoma cell lines.
  • treatment of COLO-357 and PANC-1 pancreatic cancer cells with PI-PLC abrogated selectively the mitogemc effects of FGF2 and HB-EGF in these cell lines, implying that a GPI- anchored molecule plays an essential role in FGF2- and HB-EGF- (but not IGF-1- or EGF-) mediated signaling.
  • FGF2- and HB-EGF-mitogenesis were unaffected by PI-PLC in PANC-1 cells that have been engineered to express a transmembrane- anchored form of glypican-1.
  • glypicans are the only known GPI-anchored HSPGs, and the expression levels of glypicans -2, -3, -4, or -5 are exceedingly low in pancreatic cancer cell lines.
  • endogenous glypican-1 (or another GPI- anchored molecule for which glypican-1 can substitute) is the PI-PLC-sensitive molecule that is normally required for such growth factor signaling.
  • reduction of glypican-1 protein levels in PANC-1 following expression of a glypican-1 antisense construct was associated with a marked attenuation of the mitogemc effects of FGF2 and HB-EGF in these cells, without altering EGF and IGF-1 induced mitogenesis.
  • the glypican-1 antisense clones also displayed a markedly attenuated capacity to grow in vivo.
  • the growth rate of the antisense expressing clones was considerably slower than that of parental and sham transfected PANC-1 cells.
  • this is the first demonstration that suppression of expression of any glypican genes can result in attenuated tumorigenic potential in vivo. While the mechanisms whereby inhibition of glypican-1 synthesis leads to a marked attenuation of tumor growth in vivo are not completely understood, it is well established that pancreatic cancers over-express a variety of heparin-binding growth factors.
  • pancreatic cancers express high levels of HB-EGF, FGF1, FGF2, FGF5, and HGF. It is likely therefore, that suppression of glypican-1 expression interferes with the in vivo mitogenic effects of these growth factors.
  • all of these growth factors are known to be angiogenic, and it is generally accepted that in order for tumor size to exceed 1-2 mm in vivo, cancer cells must acquire a capacity to stimulate angiogenesis.
  • decreased glypican-1 levels in the microenvironment around the cancer cells attenuates the angiogenic effects of these heparin-binding growth factors, thereby suppressing pancreatic growth indirectly.
  • HB-EGF and EGF signal by activating the same receptors, which are members of the EGF receptor family).
  • HB-EGF signaling was abrogated by PI- PLC. This observation suggests that the requirement for glypican-1 is at or upstream of the level of the receptor, precisely what one would expect for a molecule that acts by modulating or modifying growth factor-receptor interactions (i.e. a co-receptor).
  • non-GPI-anchored HSPGs e.g.
  • pancreatic carcinoma cells do not support this function in the absence of glypican-1, a result that is somewhat surprising given the fact that transfected syndecans are known to be able to confer FGF2 sensitivity upon HSPG-deficient cells. It is possible therefore, that pancreatic carcinoma cells are lacking in syndecans, or alternatively, that the co- receptor functions of glypicans and syndecans are not interchangeable. Because of the potentially widespread roles of cell surface HSPGs in growth factor signaling, it is believed to speculate that up-regulation of HSPG expression would be common in malignancies, yet this is apparently not the case.
  • glypican-1 is not up-regulated either in fibroblasts distant from the cancer cells, or in fibroblasts in chronic pancreatitis, even though this condition is associated with increased growth factor expression and the production of excessive stroma, points to an important paracrine interaction between pancreatic cancer cells and the adjacent fibroblasts.
  • glypican-1 is essential for mitogenic signaling of FGF2 and HB-EGF in pancreatic cancer cells, our findings raise the possibility that glypican-1 plays a crucial role in neoplastic transformation and tumor progression in this malignancy.
  • glypican-1 glypican-1 in breast cancer cell growth.
  • MDA-MB-231 and MDA-MB-468 both cell lines expressed glypican-1, as determined at the mRNA and protein levels.
  • PI-PLC an enzyme that cleaves GPI-anchored protein, abrogated selectively the mitogenic effects of HB-EGF and FGF-2 in both cell lines, suggesting that the mitogenic effects of these heparin-binding growth factors in breast cancer cells are dependent on the presence of surface bound glypican-1.
  • PI-PLC might remove other GPI-anchored proteins from the cell surface
  • HB-EGF, FGF-2 and HGF heparin- binding growth factors
  • EGF and IGF-1 on MDA-MB-468, only IGF-1) , which are not heparin-binding growth factors, exerted similar effects in parental, sham and glypican-1 antisense transfected cells.
  • Syndecan-1 was also expressed at high levels in some of the breast cancer samples as well as in the breast cancer cell lines. Two lines of evidence suggest that this HSPG is not as crucial as glypican-1 for the activation of mitogenic signaling by heparin-binding growth factors in breast cancer cells. First, syndecan-1 is not a GPI- anchored molecule and is therefore not removed from the cell-surface by PI-PLC treatment. Second, clones expressing the glypican-1 antisense did not exhibit a decrease in syndecan-1, yet were no longer responsive to heparin-binding growth factors.
  • glypican-1 plays a pivotal role in enhancing the growth promoting effects of heparin-binding growth factors in breast cancer cells. Therefore, pharmacological or molecular interventions that interfere with glypican-1 function or expression are useful therapeutics in breast cancer as well as in pancreatic cancer. In addition, the ability of breast cancer cells to synthesize and secrete glypican-1 at high levels show that that glypican-1 is a useful marker for this cancer both as a serum assay and as an in vitro diagnostic (illustrated in our micrographs).
  • glypican-1 and glypican-1 binding agents and suppressing agents are much the same in breast cancer, pancreatic cancer or any other glypican-1 responsive cancer (as demonstrated to be responsive by the tests described herein).
  • Therapeutic reagents operate by suppressing the glypican-1 responsive cancer by either removing the extracellular domain of the glypican (e.g., cleavage with an enzyme), binding to that domain (e.g., an antibody or drug molecule) or actually preventing the expression of that domain (as in an antisense transfection).
  • In vitro and in vivo diagnostics operate by binding to or otherwise detecting the glypican-1 molecules.
  • a reporter molecule is attached to a glypican-1 binding molecule and used to image original or metastatic cancers.
  • FGF fibroblast growth factor
  • Cerebroglycan a developmentally regulated cell-surface heparan sulfate proteoglycan, is expressed on developing axons and growth cones. Dev. Biol. 184:320-332.
  • Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions. Oncogene. 15: 1417-1424.
  • Heparin-binding EGF-like growth factor is an autocrine growth factor for rat gastric epithelial cells. Biochem. Biophys. Res. Commun. 223:36-41.
  • the division abnormally delayed (dally) gene a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila. Development. 121 :3687-3702.
  • Hepatocyte growth factor / scatter factor has distinct classes of binding site in heparan sulfate from mammary cells. Biochemistry, 37: 6003-6008, 1998.
  • Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J. Cell Biol. 135:819-27.
  • Cerebroglycan an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation. J. Cell. Biol. 124:149-160. 100 Stipp, C.S., Litwack, E.D., and Lander, A.D. Cerebroglycan : an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation. J.Cell Biol., I l l: 3165-3176, 1994.
  • K-glypican a novel GPI- anchored heparan sulfate proteoglycan that is highly expressed in developing brain and kidney. J. Cell Biol. 130:1207-18.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)

Abstract

Glypicane-1 HSPG ancré à glycosylphosphatidylinositol- (GPI-) est fortement exprimé dans le cancer du sein et du pancréas chez l'humain à la fois par les cellules cancéreuses et par les fibroblastes contigus dans le cas du cancer du pancréas, alors que l'expression de glypicane-1 est basse dans le pancréas normal et dans la pancréatite chronique. Le traitement de deux lignées cellulaires du cancer du pancréas, exprimant glypicane-1, par l'enzyme phospholipase-C spécifique pour phosphoinositide (PI-PLC) a supprimé leurs réactions mitogéniques à deux facteurs de croissance se fixant à héparine: le facteur-2 de croissance des fibroblastes (FGF2) et le facteur de croissance semblable à EGF se fixant à héparine (HB-EGF). Le traitement des cellules du cancer du sein MDA-MB-231 et MDA-MB-468 par PI-PLC a supprimé la réaction mitogénique à deux facteurs de croissance se fixant à héparine, le facteur de croissance semblable au facteur de croissance épidermique se fixant à héparine (HB-EGF) et le facteur-2 de croissance des fibroblastes (FGF-2). Syndecane-1 est également exprimé à des niveaux importants dans des tissus du cancer du sein, ainsi que dans des cellules du cancer du sein, par rapport à des tissus normaux du sein. La transfection temporaire ou permanente d'un produit génétique antisens de glypicane-1 a atténué les niveaux de protéines de glypicane-1 et la réaction mitogénique à FGF2 et HB-EGF. On peut utiliser glypicane afin de détecter le carcinome in vitro et des substances thérapeutiques qui soit se fixent (par exemple, anticorps ou médicaments) à la surface du domaine extracellulaire de glypicane-1, soit en suppriment (par exemple, enzymes) ou en empêchent l'expression (par exemple, produits génétiques antisens), sont efficaces pour retarder la croissance de carcinomes réactifs à glypicane.
EP99954963A 1998-10-16 1999-10-15 Glypicanes servant a detecter et a traiter le carcinome humain Withdrawn EP1146903A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US10451098P 1998-10-16 1998-10-16
US104510P 1998-10-16
US12162499P 1999-02-25 1999-02-25
US121624P 1999-02-25
PCT/US1999/024176 WO2000023109A1 (fr) 1998-10-16 1999-10-15 Glypicanes servant a detecter et a traiter le carcinome humain

Publications (2)

Publication Number Publication Date
EP1146903A1 true EP1146903A1 (fr) 2001-10-24
EP1146903A4 EP1146903A4 (fr) 2005-02-16

Family

ID=26801634

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99954963A Withdrawn EP1146903A4 (fr) 1998-10-16 1999-10-15 Glypicanes servant a detecter et a traiter le carcinome humain

Country Status (4)

Country Link
EP (1) EP1146903A4 (fr)
AU (1) AU769125B2 (fr)
CA (1) CA2346264A1 (fr)
WO (1) WO2000023109A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1412477A4 (fr) * 2001-07-12 2005-12-14 Exelixis Inc Glypicanes (gpc) utiles en tant que modificateurs des mecanismes de irrtk et de p21, methodes d'utilisation correspondantes
WO2004022597A1 (fr) * 2002-09-04 2004-03-18 Chugai Seiyaku Kabushiki Kaisha Anticorps d'un peptide n-terminal du gpc3 solubilise dans le sang
ATE425461T1 (de) * 2002-05-23 2009-03-15 Sunnybrook & Womens College Diagnose von hepatozellulärem karzinom
MA40764A (fr) 2014-09-26 2017-08-01 Chugai Pharmaceutical Co Ltd Agent thérapeutique induisant une cytotoxicité
ES2805085T3 (es) * 2015-01-16 2021-02-10 Glyp Holdings Pty Ltd Epítopos de glipicano y usos de éstos

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2116331T3 (es) * 1991-01-15 1998-07-16 Biotie Therapies Oy Deteccion del contenido en sindecan en los materiales biologicos tales como tejidos o fluidos fisiologicos que indican transformaciones celulares malignas.

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
INKI P ET AL: "THE ROLE OF SYNDECAN-1 IN MALIGNANCIES" ANNALS OF MEDICINE, FINNISH MEDICAL SOCIETY DUODECIM, HELSINKI, FI, vol. 28, 1996, pages 63-67, XP002927348 ISSN: 0785-3890 *
SEBESTYEN A ET AL: "Expression of Syndecan-1 in Human B Cell Chronic Lymphocytic Leukaemia" EUROPEAN JOURNAL OF CANCER, PERGAMON PRESS, OXFORD, GB, vol. 33, no. 13, November 1997 (1997-11), pages 2273-2277, XP004284563 ISSN: 0959-8049 *
See also references of WO0023109A1 *
STANLEY M. ET AL MODERN PATHOLOGY vol. 12, no. 1, January 1999, page 31A *
SU G. ET AL AM J PATHOL vol. 168, no. 6, June 2006, pages 2014 - 2026 *

Also Published As

Publication number Publication date
WO2000023109A1 (fr) 2000-04-27
AU769125B2 (en) 2004-01-15
EP1146903A4 (fr) 2005-02-16
AU1118100A (en) 2000-05-08
CA2346264A1 (fr) 2000-04-27

Similar Documents

Publication Publication Date Title
Kleeff et al. The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer.
US20070026471A1 (en) Glypican-1 in human breast cancer
Modjtahedi et al. The receptor for EGF and its ligands-expression, prognostic value and target for therapy in cancer
Matsuda et al. Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells
Berger et al. Evidence for a role of FGF‐2 and FGF receptors in the proliferation of non‐small cell lung cancer cells
US9987326B2 (en) Klotho protein and related compounds for the treatment and diagnosis of cancer
Nagane et al. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis
Okamoto et al. Expression of constitutively activated EGFRvlll in non‐small cell lung cancer
US8354228B2 (en) Human Sef isoforms and methods of using same for cancer diagnosis and gene therapy
Zhang et al. MCF-7 breast carcinoma cells overexpressing FGF-1 form vascularized, metastatic tumors in ovariectomized or tamoxifen-treated nude mice
US8007997B2 (en) Methods and compositions for inhibiting the growth of hematopoietic malignant cells
Salomon et al. The role of amphiregulin in breast cancer
Hijazi et al. Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines.
Alper et al. Anti–sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell–adhesion and tumorigenicity in ovarian cancer cells
Kleeff et al. Stable transfection of a glypican-1 antisense construct decreases tumorigenicity in PANC-1 pancreatic carcinoma cells
BRPI0707480A2 (pt) usos de um anticorpo monoclonal, um fragmento e/ou anticorpo geneticamente engenheirado ou humanizado, de uma seqüência de nucleotìdeos e de um vetor, produto, e, método para triar compostos
Normanno et al. Expression of amphiregulin, cripto-1, and heregulin-alpha in human breast-cancer cells
KR20060070491A (ko) 항종양 요법에 대한 종양 세포의 감수성 회복 및 세포 사멸유도를 위한 조성물 및 방법
Murai et al. Overexpression of c-met in oral SCC promotes hepatocyte growth factor-induced disruption of cadherin junctions and invasion
Kornmann et al. IIIc isoform of fibroblast growth factor receptor 1 is overexpressed in human pancreatic cancer and enhances tumorigenicity of hamster ductal cells
Lappi Tumor targeting through fibroblast growth factor receptors
US20030096373A1 (en) Antibodies to a novel EGF-receptor related protein (ERRP)
US10555963B2 (en) Klotho protein and related compounds for the treatment and diagnosis of cancer
AU769125B2 (en) Glypicans for the detection and treatment of human carcinoma
US8703917B2 (en) Epidermal growth factor receptor variants and pharmaceutical compositions thereof

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010426

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

A4 Supplementary search report drawn up and despatched

Effective date: 20050105

17Q First examination report despatched

Effective date: 20080124

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100504