Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin

Abstract

Up-regulation of the dolichol pathway, a "hallmark" of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF(165). Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF(165)-specific phosphotyrosine kinase activity; and (iii) Matrigel(TM) invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in Matrigel(TM) implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in Matrigel(TM) implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ∼50-60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ∼65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.

FIGURE 1.

Time course of tunicamycin inhibition of VEGF-stimulated capillary endothelial cell proliferation. A, synchronized culture of capillary endothelial cells was treated with VEGF₁₆₅ (10 ng) in the presence or absence of tunicamycin (1 μg/ml), and cell numbers were counted microscopically. VEGF₁₆₅ was added prior to the addition of tunicamycin. B, phosphotyrosine kinase activity in capillary endothelial cells. The experiment was performed per instructions from the manufacturer, and measurements were made in an automated microplate reader/EIA plate reader (Model 2550, Bio-Rad) at 450 nm. The synchronized cells were treated with tunicamycin (1 μg/ml) for 3 h and then treated with VEGF₁₆₅ for 10 min. CBO-II was added 30 min prior to the addition of VEGF₁₆₅. C, status of VEGFR1 and VEGFR2 receptors. Synchronized cells were incubated with tunicamycin for 3 h-32 h, and the levels of total VEGFR1, phospho-VEGFR1, total VEGFR2, and phospho-VEGFR2 receptors were analyzed by Western blot using anti-VEGFR1 total (1:2,000; v/v), anti-phospho-VEGFR1 (1:1,000; v/v), anti-VEGFR2 total (1:2,000; v/v), and anti-phospho-VEGFR2 (1:1,000; v/v) antibodies. Actin (1:5,000; v/v) was used as a loading control. D, quantification of VEGFR1 and VEGFR2 receptors: Densitometer scanning (arbitrary unit) of the Western blots was plotted against the time of treatment. The results are an average from three representative immunoblots for each experiment.

FIGURE 2.

Tunicamycin inhibition of VEGF-induced angiogenesis and CD34 and CD144 expression in Matrigel^TM implants. A, representative images of Matrigel^TM plugs containing heparin (left), heparin + VEGF₁₆₅ (middle), and heparin + VEGF₁₆₅ + Tunicamycin (5 μg) (right). B, H&E staining of Matrigel^TM implants (100×). Microvessels are identified with arrows. Magnified views (200×) are in insets. Histograms represent microvessels per fields (mean ± S.E. (n = 5); p < 0.001). C, immunohistochemistry of CD34 of Matrigel^TM sections (100×). The histograms are for CD34-positive cells (mean ± S.E.) per field. D, photomicrographs of immunostained CD144 from Matrigel^TM sections (100×). The histograms are for CD144-positive cells (mean ± S.E.). The numbers in each case are an average from five different slides and five regions per slide; p < 0.001.

FIGURE 3.

Thrombospondin-1 expression in Matrigel^TM implants and in capillary endothelial cells. A, quantification of TSP-1 mRNA expression in Matrigel^TM. B, quantification of TSP-1 mRNA expression in capillary endothelial cells after 3 h and 32 h of tunicamycin treatment. C left, TSP-1 protein expression in capillary endothelial cell was examined by immunoblotting (40 μg of total protein) from control and tunicamycin-treated cells (3 h and 32 h). The blot was developed with an anti-TSP-1 antibody (1:1,000; v/v) and anti-actin. C, right, histogram representing quantification of the TSP-1 protein expression as measured by Chem Doc Densitometry (Bio-Rad). The results are an average from three blots done independently.

FIGURE 4.

Tunicamycin slows down the growth of a double negative breast adenocarcinoma. The breast tumor was developed orthotopically in athymic nude mice after injecting MDA-MB-435 (ER⁻/PR⁻/EGFR⁺) human breast cancer cells and monitored by caliper measurements. The mice were treated intravenously once a week with tunicamycin (0–1.0 mg/kg). A, left, tumor growth as a function of time and tunicamycin treatment. A, right, weight of the excised tumor after 23 days. B, H&E-stained tumor sections identifying the microvascular density in control and following tunicamycin treatment. Arrows indicate microvessels. C, H&E staining identifies the mitotic index (arrowheads) in control and tunicamycin-treated breast tumor section. The histogram on the right represents the quantitative results of mitotic index averaged from ten representative areas of each tumor. The results are expressed as mean ± S.E.; p < 0.001. D, immunohistochemistry of Ki-67 and VEGF expression in breast tumor tissue sections (400×).

FIGURE 5.

Tunicamycin inhibits the growth of a triple negative breast tumor. The xenografts were developed in athymic nude mice after injecting MDA-MB-231 (ER⁻/PR⁻/EGFR⁻) human breast cancer cells and treated with tunicamycin (0.25 mg/kg) orally twice a week. A, tumor size in a control xenograft (untreated) and after tunicamycin treatment (TM-treated); B, tumor volume (mm³) as a function of time. ◆---◆, control; ■---■, tunicamycin.

FIGURE 6.

Inhibition of Matrigel^TM invasion and chemotaxis of capillary endothelial cells after tunicamycin treatment. A, synchronized culture of capillary endothelial cells either alone or after pretreating with tunicamycin (1 μg/ml) for 32 h were seeded in control and growth factor-reduced Matrigel^TM-coated transwell plates. B, cells were cultured in EMEM containing 2% fetal bovine serum in the upper chamber. Conditioned media from human breast cancer cells MCF-7 cultured in 10% fetal bovine serum along with VEGF (10 ng/ml) was used in the lower chamber as a chemo-attractant. After incubation for 24 h at 37 °C in a CO₂ incubator, the transwells were removed, cells passed through the membrane were fixed and stained with H&E. The invaded cells were quantified by counting in an optical microscope at 200× magnification and averaged after counting five fields per membrane. The histogram at the right is the quantification of cell invasion through Matrigel^TM (p < 0.001). C, image of chemotaxis of endothelial cells. Endothelial cells grown to confluence in a regular media containing 10% fetal bovine serum and switched to a media containing 0.2% fetal bovine serum for 6 h. The monolayers were scratched with a 10 μl pipette tip and cultured with or without VEGF (10 ng/ml), or tunicamycin (1 μg/ml), or tunicamycin (1 μg/ml) + VEGF₁₆₅ (10 ng/ml) for an additional 6 h. The migrated cells were quantified microscopically and the histogram at the right is the quantification of cell migration (averaging the position of the migrating cells at the wounding edges; p < 0.001); broken line indicated by →.

FIGURE 7.

Expression of cell surface glycans and induction of unfolded protein response-mediated ER stress in breast tumor microvasculature. A, WGA staining for N-glycans in microvessels from control and tunicamycin (0–1.0 mg/kg)-treated breast tumor tissue sections. Images were captured under a fluorescence microscope. B, WGA staining of tumor cells. Detection of upr-mediated ER stress in tumor microvasculature. Tumors were fixed, sectioned and stained dually with anti-CD144 (endothelial cell marker) antibody (1:50; v/v) followed by AlexaFluor 488-conjugated secondary antibody (1:100; v/v), and anti-GRP-78 antibody (1:40; v/v) followed by Rhodamin-conjugated secondary antibody (1:100; v/v). C, microvessel (arrows) from peripheral region of breast tissue section immunostained with anti-CD144 antibody (green), and D, microvessel immunostained with anti-GRP-78 antibody (red) were monitored under a fluorescence microscope. E, GRP-78 (red) staining of tumor tissue. Histology scale: 20 μm.

FIGURE 8.

Quantification of WGA staining of breast tumor microvasculature. Tumor tissue was stained with Texas-red conjugated WGA as described under “Experimental Procedures.” The fluorescence intensity of microvessels was quantified with the ImageJ program. Results are an average of vessels from five different areas in each group.