Article Figures & Data
Figures
- Figure 1.
HIP1 expression in brain tumor tissue. Tissue microarrays constructed from normal and neoplastic brain tissues were stained for HIP1 using the human monoclonal antibody HIP1/4B10. Multiple spots from each tumor were scored as follows: 0 (no staining), 1+ (low staining), 2+ (intermediate staining), and 3+ (high staining) by at least two independent observers. An overall positive score was assigned when the average score between reads was ≥2. A, glioblastoma multiforme (Glioblastoma) with all tumor cells 3+ for HIP1 staining; glioblastoma multiforme with 50% of the tumor cells 3+ for HIP1 staining; pilocytic astrocytoma 3+ for HIP1 staining; and oligodendroglioma with 1+ low staining for HIP1 except for the endothelial cells of blood vessels. B, EGFR-positive and HIP1 3+ high-grade glioblastoma multiforme. C, EGFR-negative, HIP1-negative glioblastoma multiforme. D, PDGFβR-positive, HIP1-positive, EGFR-positive, and HER3-negative glioblastoma multiforme.
- Figure 2.
Increased frequency of anti-HIP1 antibodies in sera from human brain cancer patients. Significantly more patients with brain cancer were positive for anti-HIP1 antibodies in their sera, compared with the sera of normal healthy control patients (P < 0.001), as measured by ELISA ( 15). An ELISA value >1, compared with the negative control, was considered positive. Sixty age-matched control sera samples were compared with sera from 30 glioblastoma multiforme and 10 oligodendroglioma patients. Sera from patients with melanoma, ovarian cancer, or colon cancer were not significantly more frequently positive than sera from control individuals without a cancer diagnosis. Horizontal line, median value for each group.
- Figure 3.
Association of HIP1 family members with EGFR independent of their lipid, clathrin, AP2, or actin interacting domains. A, endogenous EGFR associates with HIP1. Liver extracts from HIP1 transgenic mice were analyzed for an endogenous association between HIP1 and EGFR because the levels of EGFR in murine liver were higher than in other tissues, and human HIP1 was expressed at high enough levels to detect the endogenous interaction. Liver extract (4.9 mg) derived from a transgenic mouse that expressed human HIP1 in the liver was immunoprecipitated with either preimmune sera (lane 1) or UM323, a polyclonal antibody specific to the COOH-terminal end of HIP1 (lane 2), separated by 6% SDS-PAGE and blotted for HIP1 (monoclonal HIP1/4B10) and EGFR (sheep polyclonal, Upstate Biotechnology). B, association of HIP1 and the lipid-binding deletion mutant of HIP1 (HIP1/ΔANTH) with EGFR in vitro. Ten-centimeter dishes of 70% confluent 293T cells were transfected with 20 μg of total empty vector, EGFR, and HIP1 DNA as indicated above the blot. Thirteen hours after transfection, the cells were lysed and 1 mg of total protein was precipitated with the polyclonal anti-HIP1 antibody, UM323, or a preimmune bleed from the same rabbit as a negative control. Immunoprecipitates then were blotted for EGFR and HIP1. Lanes 1 and 3 were transfected with HIP1 and EGFR. Lanes 2 and 4 were transfected with HIP1/ΔANTH and EGFR. C, HIP1 associates with EGFR independent of clathrin or actin binding. Immunoprecipitation of transfected 293T cells, as described in (B), indicated that association of HIP1 with EGFR was dependent on transfected HIP1 (lane 6) but independent of clathrin/AP2 (ΔLD, lane 9) and actin (ΔTH, lane 10) interacting domains, as well as the lipid interacting ANTH domain. D, HIP1r associates with EGFR independent of lipid, clathrin, or actin binding. Immunoprecipitation of HIP1r-transfected 293T cells, as in (B), indicated that association of HIP1r with EGFR was independent of the HIP1r clathrin (Δ153, lane 6), lipid, or actin binding (ΔAΔT, lanes 7 and 8) domains.
- Figure 4.
EGFR interacts with the HIP1 family via overlapping regions. A, region for EGFR association with HIP1 spans HIP1 amino acids 381 to 814. Overlapping with this EGFR association region is a region (horizontal striped zone) that was previously shown to be necessary for HIP1/PDGFβR hematopoietic cell transformation ( 31). B, region for EGFR association with HIP1r spans amino acids 633 to 822. ANTH, AP180 NH2-terminal homology; CC, coiled-coil; LZ, leucine zipper; TH, talin homology.
Tables
- Table 1.
HIP1 expression in normal and neoplastic tissue samples
Tissue sample category Positive Negative Frequency PLR * Normal brain tissue (n = 75) 21 54 0.28 All brain tumors † (n = 78 total, 54 glial) 49 29 0.63 † 2.2 Glioma † (n = 7) 7 0 1.00 † 3.6 Oligodendroglioma ‡ (n = 9) 7 2 0.78 ‡ 2.8 GBM † (n = 38) 27 11 0.71 † 2.5 EGFR-positive GBM (n = 14) 9 3 0.79 2.8 PDGFβR-positive GBM (n = 6) 6 0 1.00 3.6 EGFR- or PDGFβR-positive GBM § (n = 17) 14 3 0.82 § 2.9 EGFR- and PDGFβR-negative GBM (n = 11) 5 6 0.45 1.6 -
Abbreviation: GBM, glioblastoma multiforme.
-
↵* PLR (positive likelihood ratio) = sensitivity / (1 − specificity).
-
↵† P ≤ 0.001, compared with HIP1 staining in normal brain tissue.
-
↵‡ P ≤ 0.01, compared with HIP1 staining in normal brain tissue.
-
↵§ P ≤ 0.05, compared with EGFR- and PDGFβR-negative glioblastoma multiforme.
-
- Table 2.
Frequency of a positive anti-HIP1 antibody blood test in cancer patients
Sera category Positive Negative Frequency PLR * Age (±SD), y Male (%) Normal controls 23 37 0.38 55 ± 14 44 Brain cancer † 37 3 0.93 † 2.4 51 ± 13 43 Glioblastoma † 27 3 0.90 † 2.3 54 ± 12 44 Oligodendroglioma † 10 0 1.0 † 2.6 42 ± 9 40 Melanoma 32 43 0.43 1.1 54 ± 15 56 Ovarian cancer 7 18 0.28 0.7 59 ± 13 0 Colon cancer 9 15 0.38 1.0 68 ± 10 58
Supplementary Data Bradley, et al.
Files in this Data Supplement:
Volume 67, Issue 8
