Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain - PubMed (original) (raw)

doi: 10.1002/emmm.201201623.

Aya Kobayashi, Hiroshi Okuda, Misako Watabe, Sudha K Pai, Puspa R Pandey, Shigeru Hirota, Andrew Wilber, Yin-Yuan Mo, Brian E Moore, Wen Liu, Koji Fukuda, Megumi Iiizumi, Sambad Sharma, Yin Liu, Kerui Wu, Elizabeth Peralta, Kounosuke Watabe

Affiliations

Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain

Fei Xing et al. EMBO Mol Med. 2013 Mar.

Abstract

Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL-1β which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.

Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

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Figures

Figure 1

Figure 1. Conditioned medium of brain metastatic cells up-regulates JAG1 and activates astrocytes

  1. Primary rat astrocytes were cultured in the presence of CM prepared from MB231, 231BrM, CN34 and CN34BrM cells and the expression of JAG1 was measured by qRT-PCR and Western blot (inserted photo).
  2. Primary rat astrocytes were cultured with the CM from MB231 or 231BrM, and the expression of JAG1 was measured at various time points by qRT-PCR and Western blot (inserted photo).
  3. Immortalized human astrocytes cell line (UC1) was cultured in the presence of CM from MB231 or 231BrM cells and the expression of JAG1was measured by RT-PCR.
  4. Primary rat astrocytes were cultured in the presence of CM of MB231 or 231BrM, and the expression of JAG1 and reactive astrocytes marker, GFAP, were examined by immunocytochemical staining. Bar, 100 µm. P values were calculated by a two-tailed Student's t test.

Figure 2

Figure 2. IL-1 β is highly expressed in brain metastatic cells of breast cancer

  1. CM of MB231 and 231BrM cells were subjected to cytokine array (RayBiotech) and the position of IL-1β is indicated by a red box. There are three sets of panels (A–C) and only the result of panel A was shown. The results of the other two panels were shown in Supporting Information Fig 2. Fold changes of individual cytokines that were up-regulated in the CM of 231BrM cells compared to the parental cells are listed in the right panel.
  2. The mRNA level of IL-1β in MB231 and 231BrM cells was measured by qRT-PCR. CM from MB231 and 231BrM cells was also concentrated and the amount of IL-1β was examined by Western blot (inserted photo).
  3. CM collected from MB231, 231BrM and 231BrM/shIL-1β were subjected to IL-1β ELISA assay.
  4. The mRNA level of IL-1β in CN34 and CN34BrM cells were measured by qRT-PCR. CM from CN34 and CN34BrM cells were also concentrated and the amount of IL-1β was examined by Western blot (inserted photo).
  5. Kaplan–Meier analysis for brain metastasis-free survival of 710 breast cancer patients in GEO data bases (GSE12276, GSE2034, GSE2603, GSE5327 and GSE14020). Patients were divided into two groups based on the expression status of IL-1β and IL-1α in their primary tumours.
  6. The expression of IL-1β was measured by immunohistochemical staining using the IL-1β specific antibody for breast cancer. Staining intensity of IL-1 β in primary tumours with or without brain metastasis was quantified (n = 6–11). P values were calculated by a two-tailed Student's t test.

Figure 3

Figure 3. IL-1β up-regulates JAG1 expression through NF-κB pathway in astrocytes

  1. Primary rat astrocytes were cultured in the presence of IL-1β at various doses for 24 h, and the JAG1 expression level was measured by qRT-PCR and Western blot (left panel). Primary rat astrocytes were cultured in the presence of 50 ng/ml of IL-1β, and the JAG1 expression was measured at various time points by qRT-PCR and Western blot (right panel).
  2. Primary human astrocytes were cultured in the presence of 50 ng/ml of IL-1β, and the JAG1 expression was measured by qRT-PCR and Western blot.
  3. Primary rat astrocytes were cultured in the presence of IL-1β for 24 h, and the expressions of JAG1 and GFAP were examined by immunocytochemical staining. Bar, 50 µm.
  4. Primary rat astrocytes were cultured in the presence of CM of 231BrM cells with or without 100 ng/ml IL-1RA for 24 h, and the levels of JAG1 mRNA and protein were examined by qRT-PCR and Western blot (inserted photo).
  5. Primary rat astrocytes were cultured in 231BrM CM with or without 10 µg/ml of IL-1β antibody for 24 h, and the expression of JAG1 mRNA was examined by qRT-PCR.
  6. Primary rat astrocytes were cultured in the presence or absence of IL-1β (50 ng/ml) with or without the NF-κB inhibitor, PDTC, and the expression of JAG1and P50 were measured by qRT-PCR (left panel) and Western blot (right panel). P values were calculated by a two-tailed Student's t test.

Figure 4

Figure 4. Reactive astrocytes promote self-renewal of CSCs through activation of Notch pathway

  1. Rat primary astrocytes with or without knockdown of JAG1 were grown as a monolayer, and 231BrM-GFP cells were cultured alone or on top of the astrocytes in the presence or absence of DAPT (10 µM) for 48 h. NICD expression in cancer cells was then examined by immunocytochemical staining. Bar, 100 µm.
  2. 231BrM cells were co-cultured with rat primary astrocytes for the indicated time and the population of CSCs (CD24−, CD44+, ESA+) was measured by FACS.
  3. CSCs were isolated from 231BrM cells by MACS and they were co-cultured with primary rat astrocytes, NIH3T3 or mouse brain endothelial cells (Brain ET) for 72 h. Cells were then subjected to FACS analysis using antibodies to CD24, CD44 and ESA.
  4. CSCs from 231BrM were co-cultured with rat astrocytes in the presence of various concentrations of DAPT for 72 h followed by FACS analysis using antibodies to CD24, CD44 and ESA.
  5. CSCs were isolated from 231BrM/Tet-NICD cells, and they were treated with or without tetracycline to induce NICD for 48 h followed by FACS analysis using antibodies to CD24, CD44 and ESA. P values were calculated by a two-tailed Student's t test.

Figure 5

Figure 5. Notch signalling promotes self-renewal of CSCs through up-regulation of HES5

  1. Primary rat astrocytes with or without knockdown of JAG1 were grown as a monolayer, and 231BrM-GFP cells were cultured alone or on top of the astrocytes for 48 h. GFP+ cells were then isolated by FACS, and the expression of HES5 was measured by qRT-PCR.
  2. Kaplan–Meier analysis for brain metastasis-free survival of 204 breast cancer patients (GSE12276). Patients were divided into two groups based on the expression status of HES5 in the primary tumour.
  3. HES5 mRNA levels in the primary (n = 5) and brain metastatic samples (n = 8) of breast cancer patients were examined by Taqman Real time PCR.
  4. 231BrM/Tet-NICD cells were cultured in the presence or absence of tetracycline and with or without infection of lenti virus expressing sh-HES5 for 72 h followed by FACS analysis for CSCs population.
  5. Mammosphere forming ability was measured in CSCs that were isolated from 231BrM/Tet-NICD cells in the presence or absence of tetracycline and with or without infection of sh-HES5 lenti virus. Representative photos were taken at day 18 (inserted figure). Bar, 200 µm.
  6. HES5 was ectopically expressed in 231BrM cells by lenti virus infection, and CSCs population was measured by FACS. The over expression of HES5 in 231BrM was verified by Western blot (inserted figure).
  7. CSCs were isolated from primary breast tumour cells that were infected with indicated lenti viruses, and mammosphere forming abilities were measured. Representative photos were taken at day 14 (inserted figure). Bar, 200 µm.
  8. Primary breast tumour cells with or without infection of lenti virus expressing HES5 were cultured in a low-attachment plate for 72 h followed by FACS analysis for CSCs population. P values were calculated by a two-tailed Student's t test.

Figure 6

Figure 6. Inhibition of Notch signalling and IL-1 β suppresses the metastatic growth of CSCs in vivo

  1. CSCs or non-CSCs were isolated from 231BrM cells, and a limiting dilution analysis was done by intracardially injecting various numbers of cells into nude mice followed by monitoring the incidence of brain metastasis by BLI.
  2. The brain sections of the tumour bearing mice were subjected to immunohistochemical analysis for JAG1 (red, Alexa 555), GFAP (green, FITC) and H&E staining. The astrocyes which express both JAG1 and GFAP are indicated by white arrows. Met, metastasis BP, brain peripheral, Bar, 100 µm.
  3. The brain sections of the tumour bearing mice were subjected to immunohistochemical analysis for CD44 (green, FITC), ESA (red, Alexa 555) and H&E staining. Cancer cells expressing both markers are indicated by white arrows. Bars indicate100 µm (left panel) and 50 µm(middle and right panels), respectively.
  4. CSCs were prepared from 231BrM, 231BrM/DNMAML and 231BrM/shIL-1, and 5 × 104 cells were intracardially injected into nude mice followed by monitoring tumour growth by measuring the total photon flux in the brain. Brian metastasis free survival curve was shown in right panel.
  5. CSCs were prepared from MDA231 and MDA231-IL1β, and 105 cells were intracardially injected into nude mice followed by measuring the total photon flux in the brain ex vivo at the end point (left panel). The result of brian metastasis-free survival was shown in the right panel. CSCs were prepared from 231BrM cells and 5 × 104 cells were intracardially injected to each group of mice (n = 12) followed by treatment with Compound E or vehicle only followed by measuring the total photon flux in the brain for 32 days.
  6. Proposed model for the growth of breast CSCs in the brain. IL-1β secreted from metastatic CSCs up-regulates JAG1 on the reactivated astrocytes which in turn promote self-renewal of CSCs through JAG1-Notch-HES5 axis. P values were calculated by a two-tailed Student's t test.
  7. Proposed model for the growth of breast CSCs in the brain. IL-1β secreted from metastatic CSCs up-regulates JAG1 on the reactivated astrocytes which in turn promote self-renewal of CSCs through JAG1-Notch-HES5 axis. _p_-values were calculated by a two-tailed Student's t test.

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