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Molecular Cancer Therapeutics
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Article

Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

James S. Hardwick, Yi Yang, Chunsheng Zhang, Bin Shi, Rosemary McFall, Elizabeth J. Koury, Susan L. Hill, Hongyue Dai, Robert Wasserman, Robert L. Phillips, Edward J. Weinstein, Nancy E. Kohl, Michael E. Severino, John R. Lamb and Laura Sepp-Lorenzino
James S. Hardwick
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Yi Yang
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Chunsheng Zhang
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Bin Shi
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Rosemary McFall
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Elizabeth J. Koury
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Susan L. Hill
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Hongyue Dai
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Robert Wasserman
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Robert L. Phillips
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Edward J. Weinstein
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Nancy E. Kohl
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Michael E. Severino
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John R. Lamb
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Laura Sepp-Lorenzino
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DOI: 10.1158/1535-7163.MCT-04-0209 Published March 2005
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  • Figure 1.
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    Figure 1.

    Inhibition of VEC proliferation in vitro by KDR kinase inhibitors. HDMVECs were induced to proliferate for 72 hours by growth factor addition. Cell proliferation was normalized to a control cell population that was not exposed to growth factor or KDR kinase inhibitor. nt, not treated with growth factor.

  • Figure 2.
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    Figure 2.

    Identification of a gene expression profile in proliferating vascular endothelial cells in vitro. HDMVECs were grown in culture and mitogen deprived for 24 hours as described in Materials and Methods. Magenta, up-regulated genes; cyan, down-regulated genes; black, a lack of regulation for a particular condition. Color intensity represents the fold change in regulation.

  • Figure 3.
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    Figure 3.

    Specific suppression of VEGF-induced gene expression in cultured HDMVECs. HDMVEC monolayers were stimulated to proliferate with 100 ng/mL VEGF for 24 hours in the presence or absence of KDR kinase inhibitor B. RNA populations isolated from cells exposed to VEGF or VEGF + KDR kinase inhibitor B were compared with matched control RNAs isolated from quiescent cells exposed to neither VEGF nor KDR kinase inhibitor B. Each point in the plots represents a gene sequence present on the DNA oligonucleotide microarray and is plotted according to the ratio of the two mRNA levels (experimental sample intensity/control sample intensity, Y axis) and the total mRNA quantity (experimental sample intensity + control sample intensity, X axis) for that gene. Magenta, up-regulated genes; cyan, down-regulated genes.

  • Figure 4.
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    Figure 4.

    Growth kinetics of established rat tumors following exposure to a KDR kinase inhibitor. Tumor volumes in the C6 (A) and Mat B III (B) tumor models were determined by caliper measurements. Tumors were calipered in two dimensions (length and width) and tumor volume was calculated according to the formula: (length) × (width) × (0.5 width).

  • Figure 5.
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    Figure 5.

    Identification of gene expression changes induced in rat tumors by KDR kinase inhibitors in vivo. Each row represents a distinct tumor from an individual animal. Each column represents a gene. Magenta, up-regulated genes; cyan, down-regulated genes. A, genes/sequences from rat C6 flank tumors that are regulated following 24, 48, and 72 hours of systemic exposure to the KDR kinase inhibitor KDR kinase inhibitor A. P < 0.05 for individual sequences. B, genes/sequences from rat C6 flank tumors regulated following 24, 48, and 72 hours of systemic exposure to the KDR kinase inhibitor B. P < 0.05 for individual sequences. C, genes/sequences from rat Mat B III mammary tumors regulated >1.5-fold following 100 hours of systemic exposure to the KDR kinase inhibitor A. P < 0.05 for individual sequences.

  • Figure 6.
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    Figure 6.

    Distinct tumor gene expression responses elicited by KDR inhibitors. A, Venn diagram indicating the degree of overlap between the tumor gene expression responses to KDR kinase inhibitors in C6 flank tumors and Mat B III mammary tumors. P < 0.05 for individual sequences. B, Venn diagram indicating the degree of overlap between the endothelial cell–specific tumor gene expression responses to KDR kinase inhibitors in C6 flank tumors and Mat B III mammary tumors. P < 0.05 for individual sequences. C, Venn diagram indicating the degree of overlap between the sets of endothelial cell–specific genes regulated both in vitro by mitogens and in tumor tissue by KDR kinase inhibitors. P < 0.05 for individual sequences.

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    Figure 7.

    Confirmation of microarray data by quantitative real-time PCR. Quantitative real-time PCR was done with gene-specific PCR primer pairs and amplicon-specific fluorescent probes (Taqman). A, fold changes in gene expression in tumors from KDR kinase–treated animals relative tumors from vehicle-treated animals were calculated using the ΔΔCT method (see Materials and Methods). B, mRNA levels for each gene relative the calibrator RNA pool.

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    Figure 8.

    Biomarker protein expression in rat mammary tumors is localized to vasculature. Mat B III tumor sections were incubated with antibodies against CD31 and one of the following biomarker proteins: clusterin, ANGPT2, ENDRB, or PLAU. Primary antibodies bound to the biomarker proteins and CD31 were visualized with Alexa Fluor 488–labeled and Alexa Fluor 546–labeled secondary antibodies, respectively, as described in Materials and Methods.

Tables

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  • Table 1.

    Small molecule inhibitors of KDR

    AssayKDR inhibitor A, IC50 (nmol/L)KDR inhibitor B, IC50 (nmol/L)
    KDR412
    KDR (rat)26
    FLT1124251
    FLT37279
    FLT44541
    FGFR15111,232
    FGFR2106402
    c-KIT28450
    c-Fms20835
    Platelet-derived growth factor receptor β5178
    Human umbilical vascular endothelial cell mitogenesis1731
    Rat endothelia cell mitogenesis32ND
    Molecular structureEmbedded ImageEmbedded Image
  • Table 2.

    RT-PCR primer and probe sequences

    GeneRefSeq AccPrimer sequence (or ABI Assays-on-Demand ID)
    ANGPT-2XM_225004 (Rn)Forward primer 5′-GACAGAGTCCGAATGCATGCT-3′
    Reverse primer 5′-TGCGGGTCTGGAGAAATACC-3′
    Taqman probe 5′-CCCTGTGATTCTAACCATGGCCTTCTCA-3′
    NM_001147 (Hs)Hs00169867_m1
    IFIT3XM_220059 (Rn)Forward primer 5′-TCAGGAATAGGCTGCCTGCACCCC-3′
    Reverse primer 5′-TGTGGGAGGCAACACGATTT-3′
    Taqman probe 5′-CGGTTGTTATCAGGCTCATAGGAT-3′
    FUT4NM_022219 (Rn)Forward primer 5′-GACCGAAACGTGGCTGTCTATC-3′
    Reverse primer 5′-GTGATGTGCACCGCATAGCT-3′
    Taqman probe 5′-CCGCTACTTCCACTGGCGTCGG-3′
    NM_002033 (Hs)Forward primer 5′-AATTGGGCTCCTGCACAC-3′
    Reverse primer 5′-CCAGGTGCTGCGAGTTCTC-3′
    Taqman probe 5′-TGGCCCGCTACAAGTTCTACCTGGCTT-3′
    PLAUNM_013085 (Rn)Rn00565261_m1
    NM_002658 (Hs)Hs00170182_m1
    CLUNM_012679 (Rn)Rn00562081_m1
    NM_001831 (Hs)Hs00156548_m1
    EtBNM_017333 (Rn)Rn00569139_m1
    NM_000115 (Hs)Hs00240752_m1
    GAPDHNM_017008 (Rn)4308313
    NM_002046 (Hs)402869
  • Table 3.

    Biomarkers of tumor endothelial cell proliferation

    Gene symbolRefSeq ID, H. sapiens (R. norvegicus)Gene/protein description
    ANGPT2NM_001147 (XM_344544)Angiopoietin-2. A Tie-2 ligand that functions in vascular remodeling.
    CLU (ApoJ)NM_001831 (NM_012679)Clusterin/apolipoprotein J. A secreted glycoprotein that associates with high-density lipoprotein that is implicated as both an antiapoptotic and an antiproliferative.
    ENDRB (EtB)NM_000115 (NM_017333)Endothelin receptor type B. A G protein-coupled receptor that mediates endothelin-induced vasoconstriction via the nitric oxide synthesis pathway.
    IFIT3 (GARG-49)NM_001549* (XM_220059)IFN-induced protein with tetratricopeptide repeats 3/GARG-49. Function unknown.
    FUT4NM_002033 (NM_022219)Fucosyltransferase-4. An α1,3-fucosyltransferase implicated developmental function, it is involved in the synthesis of myeloglycan, the major physiologic ligand of E-selectin. It is highly expressed in some tumors with inverse correlation to prognosis.
    PLAU (uPA)NM_002658 (NM_013085)Urokinase-type plasminogen activator. A serine-directed protease involved in vascular remodeling. It is a pro-tumor invasion and pro-metastasis factor.
    • ↵* RefSeq ID for IFIT4, the most similar human protein (60% identity and 78% similarity).

Additional Files

  • Figures
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  • Supplementary Data, Hardwick et al.

    Files in this Data Supplement:

    • Supplementary Table S1
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Molecular Cancer Therapeutics: 4 (3)
March 2005
Volume 4, Issue 3
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Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling
James S. Hardwick, Yi Yang, Chunsheng Zhang, Bin Shi, Rosemary McFall, Elizabeth J. Koury, Susan L. Hill, Hongyue Dai, Robert Wasserman, Robert L. Phillips, Edward J. Weinstein, Nancy E. Kohl, Michael E. Severino, John R. Lamb and Laura Sepp-Lorenzino
Mol Cancer Ther March 1 2005 (4) (3) 413-425; DOI: 10.1158/1535-7163.MCT-04-0209

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Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling
James S. Hardwick, Yi Yang, Chunsheng Zhang, Bin Shi, Rosemary McFall, Elizabeth J. Koury, Susan L. Hill, Hongyue Dai, Robert Wasserman, Robert L. Phillips, Edward J. Weinstein, Nancy E. Kohl, Michael E. Severino, John R. Lamb and Laura Sepp-Lorenzino
Mol Cancer Ther March 1 2005 (4) (3) 413-425; DOI: 10.1158/1535-7163.MCT-04-0209
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