1. NRP2 represses IGF-IR expression and signaling.
NRP2 represses IGF-IR expression and signaling. A, PC3 cells were either not transfected (parental) or transfected with either a GFP-shRNA (GFP-sh) or 2 independent NRP2 shRNAs (NRP2-1-sh and NRP2-2-sh) and analyzed for growth in soft agar (left graph). These transfectants (GFP-sh or NRP2-sh) were implanted in immunocompromised mice, and xenograft growth was measured on alternate days (n = 8; right graph). B, expression of IGF-IR, EGFR, and insulin receptor (IR) mRNA was quantified in PC3 transfectants (GFP-sh, NRP2-1-sh, and NRP2-2-sh) by quantitative PCR (qPCR). C, extracts from PC3 (GFP-sh or NRP2-sh) and C4-2 (GFP-sh or NRP2-sh) transfectants were immunoblotted (IB) for IGF-IR, NRP2, and actin. D, extracts from PC3 transfectants (GFP-sh or NRP1-sh) were immunoblotted for IGF-IR, NRP1, and actin. E, extracts from PC3 xenograft tumors (GFP-sh or NRP2-sh) were immunoblotted for NRP2, IGF-IR, and actin. F and G, prostate epithelial cells (p69) were transfected with either NRP2 or GFP, and extracts were immunoblotted for IGF-IR, NRP2, and actin (F). These transfectants were also analyzed for cell proliferation in response to IGF-1 using the MTT assay (middle graph). They were also serum-deprived for 12 hours and stimulated with IGF-1 (50 ng/mL) for 10 minutes. Cell extracts were used to immunoprecipitate IRS-1 and immunoblotted for phospho-tyrosine (p-Tyr) or IRS-1 (right blot). H, a significant correlation (P = 2 × 10−5) in expression of PTEN and IGF-IR was observed in human prostate specimens (n = 128) as described in Results. I, PC3 transfectants (GFP-sh, NRP2-1-sh, and NRP2-2-sh) were analyzed for anchorage-independent growth in the presence of either hIgG or an IGF-IR antibody (αIR3, 1 mg/mL). J, PC3 transfectants (GFP-sh or NRP2-sh) were implanted in immunocompromised mice. Mice were injected i.p. with either A12 (IGF-IR antibody) or IgG (40 mg/kg, 3 times a week), once the tumor volume reached approximately 150 mm3. Xenograft growth was measured on alternate days. Two groups of mice (NRP2-sh+ hIgG and NRP2-sh+ A12) exhibited significant inhibition of tumor growth compared with the remaining 2 groups (GFP-sh, hIgG and GFP-sh, A12). *, P < Eight tumors were analyzed for each group.
Hira Lal Goel et al. Cancer Discovery 2012;2:
©2012 by American Association for Cancer Research