Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells

Abstract

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In this study, we aimed to define the mechanism of Notch-ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch ligands in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly upregulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a γ-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting Akt phosphorylation. Importantly, Jagged2 was also found to be upregulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma has a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer.

Fig. 1. Jagged2 expression is correlated with overall and metastasis free- survival of breast cancer patients

(A) Univariate survival analyses by log-rank were conducted to assess the contribution of the Notch ligands to disease prognosis in 779 patients using GEO database (GSE7390, GSE2034, and NKI-295). (B) Metastasis free- and overall- survival rates over a period of 5 years were analyzed in 295 patients (NKI-295) with breast cancer, in relation to Jagged2 expression. Solid line, Jagged2 negative patients; dotted line, patients with increased expression of Jagged2. (C) Multivariate Cox regression analysis of GSE7390 was conducted to assess the contribution of the indicated variables to disease prognosis. b, the estimation for the regression coefficient β; CI, confidence interval.

Fig. 2. Expression of cleaved Notch1 (NICD), Jagged2 and hypoxia markers at invasive front of breast tumor tissue

(A) Representative photos of breast tumor specimens at various stages after IHC with anti-NICD antibody. (B) Staining intensity of NICD at different stages was quantified. (C) Representative photos of two consecutive sections stained with anti-NICD and anti-Jagged2 antibodies at invasive front and inside of tumor. (D) Representative photos of IHC using antibodies to NICD, CA9 and Glut1 at invasive front and inside of tumor.

Fig. 3. Hypoxia significantly augments the expression of Jagged2

(A) Breast cancer cell lines, MCF7 and MDA-MB231, were cultured in two sets of 24-well plates under normoxic (21%O₂) or hypoxic (1%O₂) conditions for 24 hrs. One set of cells (in triplicate) was collected, and RNA was prepared. The samples were then subjected to qRT-PCR using primers for the Jagged2 and β-actin genes. Another set of cells was collected, and the cell lysates were subjected to Western blot analysis using anti-Jagged2 and anti-tubulin antibodies (inserted fig). For Jagged2 reporter assay, MCF7 cells were transfected with the Jagged2 reporter plasmid and they were incubated under normoxia or hypoxia. Luciferase activity was measured after 24hrs incubation. (B) MCF7 cells were transfected with Notch reporter plasmid (4XCBF-Luc) and they were incubated under normoxia or hypoxia for 24 hrs followed by luciferase activity assay. Western blot was performed to measure the protein level of NICD and HIF1α in MCF7 cells under normoxia or hypoxia (inserted figure). (C) MDA-MB231 were cultured under 1%O₂ and NICD immunocytochemistry was performed. Values are means ± SD of triplicate measurements. *, P<0.01.

Fig. 4. Notch signaling promotes hypoxia-induced cell survival and EMT

(A) mRNA and whole cell lysates were prepared from the non-tumorigenic cell line, MCF10A and three human breast cancer cell lines, MCF7, MDA231 and MDA231LM, and quantitative RT-PCR and Western blot were performed to evaluate the amount of Jagged2 expression. (B) MDA231, MDA231LM and MDA231LM-DNMAML were cultured in 96-well plates under hypoxic condition (1%O₂). Cell viability was measured at different time point by MTT assay. (C) MDA231 and MDA231LM cells were infected with sh-Jagged2 and pSin-Jagged2 lentivirus, respectively. Cells were also infected with control lentiviruses. These cells were incubated for 12 hours under normoxic condition and they were transferred to hypoxic condition (1%O₂). After 72 hrs, cell viability was measured by MTT assay. Knock-down and ectopic expression of Jagged2 were also examined by Western blot (inserted photos). (D) MDA231LM cells were also cultured in the presence or absence of DAPT under hypoxia for 48 hrs and the expression of phospho-Akt was examined by Western blot. (E) MDA231LM cells were seeded in Matrigel invasion chambers in the presence or absence of DAPT (20 µM) under hypoxia. After 24hrs incubation, the number of invading cells was counted. (F) mRNA were prepared from MCF7, MDA231 and MDA231LM, and qRT-PCR was performed to evaluate the snail expression.(G) MDA231LM cells were cultured in the presence or absence of DAPT under normoxia or hypoxia for 48 hrs and cell morphology was observed under microscope. (H) mRNA levels of E-cadherin expression were examined in MDA231LM (left panel) and MCF7 cells (right panel) that were cultured under normoxia or hypoxia with or without the treatment of DAPT (20 µm). (I) MDA231LM cells were infected with sh-Jagged2 or control lentivirus for 12hrs under normoxia, and they were transferred to hypoxic condition (1%O₂). After 48 hrs of incubation, mRNA levels of Hes1 and snail were measured by qRT-PCR. Values are means ± SD of triplicate measurements. *, P<0.01.

Fig. 5. Hypoxia up regulates Jagged2 expression in bone marrow stromal cells

(A) Human bone marrow stromal cell line, HS5, was cultured in two sets of 24-well plates under normoxic (21%O₂) or hypoxic (1%O₂) conditions for up to 48hrs. One set of cells was collected and RNA was prepared. The samples were then subjected to qRT-PCR using primers for the Jagged2 and β-actin genes. Another set of cells was collected, and the cell lysates were subjected to Western blot analysis using anti-Jagged2 and anti-tubulin antibodies (inserted figure). Similar experiments were performed to examine the mRNA and protein levels of Jagged2 in HS5 after DFO treatment by qRT-PCR and Western blot (right panel). (B) MDA231BoM cells which stably expressed the Notch reporter gene were seeded on monolayer of HS5 cells followed by DFO (left panel) or hypoxia (right panel) treatment. Luciferase activity was measured after 48hrs of co-culture. Values are means ± SD of triplicate measurements. *, P<0.01.

Fig. 6. Notch signaling promotes self-renewal of cancer stem-like cells under hypoxia

(A) Stem-like cells (CD24⁻CD44⁺ESA⁺) were isolated from MDA231BoM/GFP and they were co-cultured with HS5 monolayer under normoxia and hypoxia for 48hrs. Immunocytochemistry was performed using anti-NICD antibody. (B) MDA231BoM/GFP stem-like cells were co-cultured with HS5 monolayer for 48hrs under normoxia and hypoxia in the presence or absence of DAPT (20 µm). Cells were then subjected to FACS analysis by using anti-NICD antibody. (C) 231BoM/GFP stem-like cells were co-cultured with HS5 monolayer under normoxia and hypoxia in the presence or absence of DAPT (20 µm) for 72hrs. The number of CD24⁻CD44⁺ESA⁺ cells was measured by incubating cells with anti-CD24, anti-CD44 and anti-ESA antibodies followed by FACS analysis. (D) MDA231BoM-Tet/NICD cells were cultured in the presence or absence of tetracycline for 48 hrs and cells were subjected to FACS analysis by using anti-CD24, anti-CD44 and anti-ESA antibodies. Values are means ± SD of triplicate measurements. *, P<0.01.