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Genes that mediate breast cancer metastasis to the brain - PubMed

  • ️Thu Jan 01 2009

. 2009 Jun 18;459(7249):1005-9.

doi: 10.1038/nature08021. Epub 2009 May 6.

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Genes that mediate breast cancer metastasis to the brain

Paula D Bos et al. Nature. 2009.

Abstract

The molecular basis for breast cancer metastasis to the brain is largely unknown. Brain relapse typically occurs years after the removal of a breast tumour, suggesting that disseminated cancer cells must acquire specialized functions to take over this organ. Here we show that breast cancer metastasis to the brain involves mediators of extravasation through non-fenestrated capillaries, complemented by specific enhancers of blood-brain barrier crossing and brain colonization. We isolated cells that preferentially infiltrate the brain from patients with advanced disease. Gene expression analysis of these cells and of clinical samples, coupled with functional analysis, identified the cyclooxygenase COX2 (also known as PTGS2), the epidermal growth factor receptor (EGFR) ligand HBEGF, and the alpha2,6-sialyltransferase ST6GALNAC5 as mediators of cancer cell passage through the blood-brain barrier. EGFR ligands and COX2 were previously linked to breast cancer infiltration of the lungs, but not the bones or liver, suggesting a sharing of these mediators in cerebral and pulmonary metastases. In contrast, ST6GALNAC5 specifically mediates brain metastasis. Normally restricted to the brain, the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier. This co-option of a brain sialyltransferase highlights the role of cell-surface glycosylation in organ-specific metastatic interactions.

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Figures

Figure 1
Figure 1. Isolation and characterization of brain metastatic variants

a, b, Flowcharts of the in-vivo-selected brain metastatic derivatives, and Kaplan–Meier survival curves for brain metastasis-free survival of representative CN34 (parental n = 8, BrM2c n =14, BrM2a n = 3) (a) and MDA231 (parental n = 7, LM n = 7, BoM n = 7, BrM2a n = 23) (b) cell line variants. A log-rank test was used to compare the survival curves of each cell line to the parental line. BoM, BrM and LM indicate bone, brain and lung metastatic derivative, respectively. c, Bioluminescence image of a mouse with brain and leptomeningeal metastasis by CN34-BrM2c cells. d, Magnetic resonance imaging (MRI) of a brain metastatic lesion (dashed line) showing a hemorrhagic core, and brain oedema (arrowheads). e, f, Representative haematoxylin and eosin (H&E)-stained sections of a mouse brain containing a CN34-BrM2c lesion (original magnification, ×2 (e) and ×10 (f)). g, H&E staining of a section showing MDA231-BrM2a cell colonization of the dorsal (DM) and ventral (VM) meninges. ID, intervertebral disc; SC, spinal cord; VB, vertebral body. Original magnification, ×5. h, MDA231-BrM2a brain metastatic lesion showing reactive glia (RG, arrowheads) around the metastatic lesion (Met). Tumour cells express green fluorescent protein (GFP), and glial cells are stained with the glial marker glial fibrillary protein (GFAP, purple). BP, brain parenchyma; DAPI, 4,6-diamidino-2-phenylindole. i, GPF+ MDA231-BrM2a cells (arrowheads) arrested in brain capillaries (red, rhodamine dextran) 24 h after intracardiac injection into mice. Nuclei were stained with DAPI (blue).

Figure 2
Figure 2. COX2 and EGFR ligands as mediators of brain metastasis and BBB transmigration

a, b, Kaplan–Meier curves for brain metastasis-free survival on the basis of BrMS status in an independent cohort of 192 breast tumours (a), and in a combined cohort of 262 breast tumours from patients who received no adjuvant therapy (b). c, Kaplan–Meier curves for brain metastasis-free survival of mice injected with the indicated cell lines expressing short hairpin RNA (shRNA) vector control or shRNA targeting COX2. d, Kaplan–Meier curves for brain metastasis-free survival of mice injected with the indicated cell lines and treated with cetuximab or vehicle control. e, Schematic of the in vitro BBB model assay system. HUVEC, human umbilical vein endothelial cells. f, In vitro BBB transmigration activity of the indicated cell lines and conditions. The number of transmigrated cells relative to the parental cell lines is plotted. Error bars, s.e.m.; n = 6–20. P values were determined by log rank test (a–d) and one-tailed unpaired t-test (f).

Figure 3
Figure 3. ST6GALNAC5 expression and activity in brain metastasis from breast cancer

a, Quantification of SNA staining in mammary fat pad tumours formed by parental CN34 or CN34-BrM2c cells in mice. a.u., arbitrary units. b, SNA staining of a mouse brain metastasis after intracardiac inoculation of MDA231-BrM2a cells. Scale bar, 50 μm c, SNA staining of representative human brain and lung metastases samples from the same breast cancer patient. Scale bars, 20μm. d, Distribution of SNA staining intensity, quantified by Metamorph analysis, in 12 brain and 11 lung metastases resected from breast cancer patients. P values (a, d) were determined by Mann–Whitney one-tailed test. e, Heat map showing the relative ST6GALNAC5 expression levels in a panel of 13 brain, 8 bone, 3 liver, 12 lung and 2 ovary human metastases from breast cancer patients. Included for comparison are the parental (par) and brain metastatic derivatives from MDA231 and CN34 cells. Data are on the basis of Affymetrix probe intensity.

Figure 4
Figure 4. The brain-specific sialyltransferase ST6GALNAC5 as a BBB extravasation and brain metastasis gene

a, In vitro BBB transmigration activity of the indicated cell lines. Ctrl, control; par, parental; ST6, ST6GALNAC5. Error bars, s.e.m.; n = 9–27; P values were determined by Mann–Whitney one-tailed test. b, Kaplan–Meier curves for brain metastasis-free survival of mice injected with CN34-BrM2 cells expressing an shRNA targeting ST6GALNAC5 or an empty vector control, and then treated with cetuximab or vehicle. P values were determined using log-rank test. c, In vitro BBB transmigration activity of LM2 cells transduced with an empty vector or with ST6GALNAC5. Error bars, s.e.m.; n = 22–27; P values were determined by Mann–Whitney one-tailed test. d, Anti-GFP immunostaining of representative lesions from mice injected intracardially with the indicated cell lines. Red arrowheads show individual tumour foci; original magnification, ×10. e, Schematic model of organ-specific metastatic extravasation of breast cancer cells. Extravasation into the bone marrow is a relatively permissive process owing to the fenestrated endothelium lining the sinusoid capillaries. Extravasation into the pulmonary or brain parenchyma requires specific functions for breaching the non-fenestrated capillary walls of these organs. Shared mediators of extravasation include, among others, COX2 and EGFR ligands such as epiregulin and HBEGF. Passage through the BBB requires further mediators including, but not limited to, the brain-specific sialyltransferase ST6GALNAC5. Competence to colonize each organ requires additional mediators.

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