A therapeutic target for prostate cancer based on angiogenin-stimulated angiogenesis and cancer cell proliferation

Abstract

Human angiogenin is progressively up-regulated in the prostate epithelial cells during the development of prostate cancer from prostate intraepithelial neoplasia (PIN) to invasive adenocarcinoma. Mouse angiogenin is the most up-regulated gene in AKT-induced PIN in prostate-restricted AKT transgenic mice. These results prompted us to study the role that angiogenin plays in prostate cancer. Here, we report that, in addition to its well established role in mediating angiogenesis, angiogenin also directly stimulates prostate cancer cell proliferation. Angiogenin undergoes nuclear translocation in PC-3 human prostate cancer cells grown both in vitro and in mice. Thus, knocking down angiogenin expression in PC-3 human prostate adenocarcinoma cells inhibits ribosomal RNA transcription, in vitro cell proliferation, colony formation in soft agar, and xenograft growth in athymic mice. Blockade of nuclear translocation of angiogenin by the aminoglycoside antibiotic neomycin inhibited PC-3 cell tumor growth in athymic mice and was accompanied by a decrease in both cancer cell proliferation and angiogenesis. These results suggest that angiogenin has a dual effect, angiogenesis and cancer cell proliferation, in prostate cancer and may serve as a molecular target for drug development. Blocking nuclear translocation of angiogenin could have a combined benefit of antiangiogenesis and chemotherapy in treating prostate cancer.

Fig. 1.

IHC staining of human angiogenin in prostate tissue samples. Prostate tissue samples from normal (A), BPH (B), and cancer (C) patients were stained with mAb 26-2F (30 μg/ml) and visualized with Dako's Envision kit. (A) Weak staining was observed in the stroma in the normal prostate tissue. (B and C) Enhanced staining was observed in BPH (B) and prostate cancer (C) tissues, with strong cytoplasmic and nuclear staining. (Magnification: ×400.)

Fig. 2.

Down-regulation of angiogenin in PC-3 cells inhibits rRNA transcription and cell proliferation. PC-3 cells were transfected with an angiogenin RNAi plasmid, pANG-RNAi, or with the vector control pBS/U6. Stable transfectants were selected with 0.5 μg/ml puromycin for 2 weeks. (A) Secreted angiogenin levels determined by ELISA. (B) The steady-state level of 47S rRNA determined by Northern blotting with actin mRNA as the loading control. (C) Cell proliferation as determined with a Coulter counter. When present, exogenous angiogenin was 1 μg/ml.

Fig. 3.

Knocking down angiogenin expression in PC-3 cells decreases tumorigenicity. (A) Soft agar assay in which cells were seeded at a density of 4 × 10³ cells per 35-mm dish and cultured in 0.35% soft agar in DMEM plus 10% FBS at 37°C for 7 days. When present, angiogenin was added to both the soft agar and the medium at 0.1 μg/ml. The colonies were stained with 0.05% crystal violet. Colony numbers in the entire dish were counted. The average colony size was determined by measuring the diameters of colonies in 10 microscope fields with a microcaliper. (B and C) Xenograft growth of PC-3 tumors in nude mice. The vector control (pBS/U6) and the angiogenin RNAi (pANG-RNAi) transfectants (1 × 10⁶ cells per mouse) were injected s.c. (eight mice per group) into 6-week-old male athymic mice. (B) Mice were checked daily for tumor appearance by palpation, and tumor volume was measured every 3 days. (C) Tumors were removed on day 31 and weighed.

Fig. 4.

IHC staining of angiogenin, PCNA, and neovessels. Thin sections (4 μm) from formalin-fixed, paraffin-embedded tumor tissues derived from vector-transfected PC-3 cells (A, C, and E) and from angiogenin RNAi-transfected PC-3 cells (B, D, and F) were stained with antiangiogenin (A and B), anti-PCNA (C and D), and anti-VWF (E and F) antibodies. The bound primary antibodies were visualized with Dako's Envision system. VWF-positive vessels in each tumor were counted in the five most vascularized areas at ×200 magnification, and the numbers were averaged. Vessel density (vessels per field) is shown as mean ± SD for each group. PCNA-positive and total numbers of cells were counted in five randomly selected areas at ×200 magnification. Images shown were from a representative animal of each group.

Fig. 5.

Effect of neomycin on PC-3 cell tumor growth in athymic mice. (A and B) Inhibition of nuclear translocation of angiogenin. PC-3 cells were cultured in DMEM plus 10% FBS for 24 h and then incubated with 1 μg/ml angiogenin in the absence (A) or presence (B) of 100 μM neomycin at 37°C for 30 min. Angiogenin was visualized with 26-2F and Alexa Fluor 488-labeled goat anti-mouse IgG. (C–H) Inhibition of tumor growth. PC-3 cells (5 × 10⁵ in 67 μl of Hanks' balanced salt solution) were mixed with 33 μl of Matrigel. The mixture was injected into the left shoulder of the mice. The mice then received s.c. injections of neomycin at a dose of 60 mg/kg of body weight or PBS daily for 2 weeks, followed by injections every other day for another 6 weeks. Twelve mice were used per group. (C) Mice were examined daily by palpation for tumor appearance. (D) At day 56, mice were killed, and tumor tissues were removed and weighed. (E and F) Tissue specimens were fixed in 10% formalin, and 4-μm paraffin sections were cut. Proliferating cells were stained with an anti-PCNA mAb. PCNA-positive cells and total numbers of cells were counted in five randomly selected areas at ×200 magnification. (G and H) Neovessels were stained with an anti-VWF antibody, and neovessels in each tumor were counted in the five most vascularized areas at ×200 magnification.