uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells

Abstract

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. In this study, we show that breast cancer cells cultured in 1.0% O(2) demonstrate changes consistent with epithelial-mesenchymal transition (EMT). Snail translocates to the nucleus, and E-cadherin is lost from plasma membranes. Vimentin expression, cell migration, Matrigel invasion, and collagen remodeling are increased. Hypoxia-induced EMT is accompanied by increased expression of the urokinase-type plasminogen activator receptor (uPAR) and activation of cell signaling factors downstream of uPAR, including Akt and Rac1. Glycogen synthase kinase-3beta is phosphorylated, and Snail expression is increased. Hypoxia-induced EMT is blocked by uPAR gene silencing and mimicked by uPAR overexpression in normoxia. Antagonizing Rac1 or phosphatidylinositol 3-kinase also inhibits development of cellular properties associated with EMT in hypoxia. Breast cancer cells implanted on chick chorioallantoic membranes and treated with CoCl(2), to model hypoxia, demonstrate increased dissemination. We conclude that in hypoxia, uPAR activates diverse cell signaling pathways that cooperatively induce EMT and may promote cancer metastasis.

Figure 1.

Hypoxia induces EMT in MDA-MB-468 cells. (A) Cells were cultured for 50 h in 21% O₂ (normoxia; N) or 1.0% O₂ (hypoxia; H). Cell images were captured by phase-contrast microscopy. Cell extracts were subjected to SDS-PAGE and immunoblot analysis to detect vimentin (V), and tubulin (T). (B–D) Cells that were incubated for 50 h in 21% O₂ (N) or 1.0% O₂ (H) were immunostained to detect E-cadherin (B), Snail (C), or vimentin (D), in the green channel, with phalloidin (red), and with DAPI (blue). Bar, 50 μm.

Figure 2.

Hypoxia promotes cell migration, invasion, and collagen remodeling. (A) MDA-MB-468 cells were added to Transwell chambers, in which the membranes were precoated with FBS (cell migration; n = 21) or in which reconstituted Matrigel was present (cell invasion; n = 10). Studies were performed for 24 h in 21% O₂ (gray bar) or 1.0% O₂ (black bar). Migration and invasion are expressed relative to the levels observed with normoxic controls (mean ± SEM). (B) MDA-MB-468 cells were treated with CoCl₂ or with vehicle. Cells were allowed to migrate for 24 h. Cell migration is expressed relative to that observed with vehicle (mean ± SEM; n = 3). *, P < 0.05; **, P < 0.005. (C) Cells were cultured for 18 h on fluorescein-labeled type I collagen in 21% (normoxia) or 1.0% O₂ (hypoxia). Type I collagen remodeling was assessed. Cell nuclei are stained with DAPI. Bar, 50 μm.

Figure 3.

Hypoxia increases uPAR levels and activates cell signaling factors known to be downstream of uPAR. (A) MDA-MB-468 cells were cultured for 24 h in 21% O₂ (N) or 1.0% O₂ (H). Cell extracts were subjected to SDS-PAGE and immunoblot analysis to detect human uPAR using antibody 3932 and tubulin. (B) MDA-MB-468 cells were cultured for 15 h in 21% O₂ (N) or 1.0% O₂ (H), treated with 100 μM CoCl₂ or with vehicle (control; C). Cell extracts were affinity precipitated with PAK-1 PBD and subjected to immunoblot analysis to detect GTP-bound Rac1. The original cell extracts were studied by immunoblot analysis to determine total Rac1. Cell extracts were also probed for phosphorylated ERK/MAPK and HIF-1α. (C) The results of three separate experiments were averaged to determine the percentage of increase in activated Rac1 in hypoxia (mean ± SEM; n = 3).

Figure 4.

uPAR is necessary for hypoxia-promoted cell migration, invasion, and Rac1 activation. (A) MDA-MB-468 cells were pretreated with 25 μg/ml uPA-specific antibody 3471 and 25 μg/ml uPAR-specific antibody 399R or with 50 μg/ml control IgG. Cells were allowed to migrate in Transwell chambers for 24 h in 21% O₂ (gray bars) or 1.0% O₂ (black bars). Cell migration is expressed as a percentage of that observed with control IgG in normoxia (mean ± SEM; n = 6). (B) MDA-MB-468 cells expressing empty vector (pSUPER), sh-uPAR6 cells, and sh-uPAR12 cells were cultured for 24 h in 21% O₂ (gray bars) or 1.0% O₂ (black bars). uPAR mRNA was determined by qPCR. Results are normalized against HPRT-1 and compared with the level observed in the pSUPER cells in normoxia (mean ± SEM; n = 3). (C) pSUPER, sh-uPAR6, and sh-uPAR12 cells were allowed to migrate in Transwell chamber (cell migration) or to invade Matrigel (cell invasion) for 24 h in 21% O₂ (gray bars) or 1.0% O₂ (black bars). Results are expressed as a percentage of that observed with pSUPER cells in normoxia (mean ± SEM; n = 7 and n = 6, respectively). (D) pSUPER, sh-uPAR6, and sh-uPAR12 cells were cultured for 15 h in 21% O₂ (N) or in 1.0% O₂ (H). Cell extracts were affinity precipitated with PAK-1 PBD and subjected to immunoblot analysis to determine GTP-bound Rac1. The original extracts were subjected to immunoblot analysis for tubulin, as a loading control.

Figure 5.

uPAR is necessary for hypoxia-induced EMT and collagen remodeling. (A) pSUPER, sh-uPAR6, and sh-uPAR12 cells were cultured for 50 h in 21% O₂ (N) or 1.0% O₂ (H). The preparations were immunostained for E-cadherin (green channel), phalloidin (red channel), and DAPI. (B) pSUPER, sh-uPAR6, and sh-uPAR12 cells were cultured for 18 h on coverslips precoated with fluorescein-labeled type I collagen in 21% O₂ (N) or 1.0% O₂ (H). Cell nuclei are stained with DAPI. Bars, 50 μm.

Figure 6.

Rac1 is necessary for hypoxia-induced cell migration, invasion, and collagen remodeling. (A) MDA-MB-468 cells were transfected to express DN-MEK1 and GFP or GFP alone. Cells were allowed to migrate for 24 h in Transwell chambers (cell migration) or in chambers with reconstituted Matrigel (cell invasion). Experiments were performed in 21% O₂ (gray bars) or 1.0% O₂ (black bars). Migration and invasion are compared with the levels observed with normoxic control cells that expressed GFP (mean ± SEM; n = 3). (B) MDA-MB-468 cells were treated with 100 μM CoCl₂ (black bars) or with vehicle (gray bars). The same cells also were treated with 50 μM PD098059 or vehicle (control), as indicated. Transwell migration proceeded for 24 h. Cell migration is expressed relative to that observed with cells treated with vehicle (mean ± SEM; n = 3). (C) MDA-MB-468 cells were transfected to express DN-Rac1 and GFP or GFP alone. Cells were allowed to migrate for 24 h in Transwell chambers (cell migration) or in chambers with reconstituted Matrigel (cell invasion) in 21% O₂ (gray bars) or 1.0% O₂ (black bars). Results are expressed relative to that observed in normoxic control cells that expressed only GFP (mean ± SEM; n = 6). (D) Cells were transfected to express DN-Rac1 and GFP or GFP alone and cultured for 18 h on collagen labeled with Alexa Fluor 594 in 21% O₂ (normoxia) or 1.0% O₂ (hypoxia). Nuclei are stained with DAPI. Bar, 50 μm.

Figure 7.

uPAR-dependent activation of the PI3K–Akt pathway is necessary to induce EMT in hypoxia. (A and B) MDA-MB-468 cells were cultured for 15 h in 21% O₂ (N) or 1.0% O₂ (H). (A) Cell extracts were subjected to immunoblot analysis to detect phosphorylated Akt (p-Akt), phosphorylated GSK-3β (p-GSK-3β), Snail, and tubulin. (B) Snail mRNA was determined by qPCR (mean ± SEM; n = 4). (C) MDA-MB-468 cells were treated with 50 μM synthetic peptide (α325) or 50 μM scrambled peptide (scα325) for 15 h in 21% O₂ (N) or 1.0% O₂ (H). Cell extracts were subjected to immunoblot analysis to detect phosphorylated Akt and tubulin. (D) MDA-MB-468 cells were treated with10 μM of the PI3K inhibitor LY294002 or with vehicle for 15 h in 21% O₂ (normoxia) or 1.0% O₂ (hypoxia). Cell extracts were subjected to immunoblot analysis to detect Snail and tubulin (representative of three studies). All lanes are from the same immunoblot (same exposure). (E–G) MDA-MB-468 cells were treated with 10 μM LY294002 or vehicle (control) and cultured for 50 h in 21% O₂ (normoxia; N) or 1.0% O₂ (hypoxia; H). (E) Cell images were captured using phase-contrast microscopy. (F) Cells were immunostained to detect E-cadherin (green). The same cells were stained with phalloidin (red) and DAPI. (G) Snail mRNA was determined by qPCR. Results are compared with the level observed in control cells in normoxia (mean ± SEM; n = 3). Bar, 50 μm.

Figure 8.

uPAR overexpression is sufficient to induce EMT under normoxia. (A) MDA-MB-468 cells were cotransfected to transiently express GFP and uPAR (uPAR) or empty vector (pcDNA). Cells were allowed to migrate in Transwell chambers (cell migration; n = 9) or invade Matrigel (cell invasion; n = 8) for 24 h. Results are expressed as a percentage of that observed with normoxic GFP-expressing control cells (mean ± SEM). (B) Extracts from cells transfected with empty vector, uPAR-overexpressing C14 cells, and uPAR-overexpressing C18 cells were subjected to immunoblot analysis to detect uPAR and total ERK/MAPK, as a loading control. Equivalent cell extracts were probed to detect E-cadherin, vimentin, and total ERK/MAPK. All lanes are from the same immunoblot (same exposure). (C) pcDNA, C14, and C18 cells were cultured in 21% O₂. Representative images were captured by phase-contrast microscopy. (D) pcDNA, C14, and C18 cells were immunostained to detect E-cadherin (green channel). The same cells also were stained with phalloidin (red channel) and DAPI. Bars, 50 μm.

Figure 9.

CoCl₂ treatment promotes MDA-MB-468 cancer cell dissemination from CAMs. GFP-expressing MDA-MB-468 cells were inoculated onto CAMs at 9 d. Tumors were allowed to develop for 11 d. The cells on the CAMs were treated daily with 25 μl of 100 μM CoCl₂ (black bars) or vehicle (gray bars). (A) Primary tumors were photographed on a stereomicroscope. (B) GFP-expressing cells were imaged by fluorescence microscopy. (C) Tumors from some eggs were harvested 4 d after inoculation. RNA was isolated and analyzed by qPCR to determine levels of VEGF and uPAR mRNA. mRNA levels were standardized against the levels present in vehicle-treated tumors (mean ± SEM; n = 3). (D) Chick embryos were harvested 11 d after inoculation of tumor cells on the CAMs. The number of GFP- expressing cells/cell clusters in the heart–lung block was determined by fluorescent microscopy (mean ± SEM; n = 9).

Figure 10.

uPAR expression and EMT in hypoxic cancer cell lines. (A) MDA-MB-468 (468), MCF-7, MDA-MB-231 (231), MDA-MB-435 (435), SK-BR3, A431, ZR-75-1 (ZR75), and SCC15 (SCC) cells were cultured in 21% O₂ (gray bars) or 1.0% O₂ (black bars). uPAR mRNA was determined by qPCR and standardized against the level present in MDA-MB-468 cells under normoxia (mean ± SEM; n ≥ 3). *, P < 0.05. (B) ZR-75-1 and SCC15 cells were cultured in 21% O₂ (normoxia) or 1.0% O₂ (hypoxia). Images were captured using phase-contrast microscopy. Cell extracts were subjected to immunoblot analysis to detect vimentin (V) and tubulin (T). Bars, 50 μm.