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. 2014 Jan 1;20(1):56-67.
doi: 10.1158/1078-0432.CCR-13-1255. Epub 2013 Oct 16.

αB-crystallin: a novel regulator of breast cancer metastasis to the brain

Dmitry Malin  1 , Elena Strekalova, Vladimir Petrovic, Allison M Deal, Abraham Al Ahmad, Barbara Adamo, C Ryan Miller, Andrey Ugolkov, Chad Livasy, Karen Fritchie, Erika Hamilton, Kimberly Blackwell, Joseph Geradts, Matt Ewend, Lisa Carey, Eric V Shusta, Carey K Anders, Vincent L Cryns
Affiliations

αB-crystallin: a novel regulator of breast cancer metastasis to the brain

Dmitry Malin et al. Clin Cancer Res. .

Abstract

Purpose: Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone αB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of αB-crystallin in brain metastasis in TNBCs.

Experimental design: αB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among patients with breast cancer with brain metastases. αB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte coculture blood-brain barrier (BBB) model were examined. In addition, the effects of overexpressing or silencing αB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models.

Results: In a cohort of women with breast cancer brain metastasis, αB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among patients with TNBC. Stable overexpression of αB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing αB-crystallin inhibited these events. αB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through an α3β1 integrin-dependent mechanism. αB-crystallin overexpression promoted brain metastasis, whereas silencing αB-crystallin inhibited brain metastasis in orthotopic TNBC models.

Conclusion: αB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. αB-crystallin expression in primary breast tumors is associated with poor survival in patients with breast cancer brain metastases
(A) Representative IHC staining of paired breast tumors and brain metastases from two patients. (B) Kaplan Meier survival curves by αB-crystallin expression (positive in red and negative in blue) for all breast cancer patients (upper panels) and for patients with TNBC (lowers panel). Overall survival (OS) is plotted in the left-hand panels and survival after breast cancer brain metastasis (BCBM) is shown in the right-hand panels.
Figure 2
Figure 2. αB-crystallin overexpression in TNBC cells enhances adhesion to brain endothelium, transendothelial migration, and BBB transmigration in vitro
(A) Immunocytochemistry analysis of confluent primary HBMEC (passage #4–5) monolayers. The tight junction proteins claudin-5, occludin and ZO-1 were expressed at varied levels throughout the monolayer. HBMEC clusters with elevated expression and appropriate localization of these proteins are shown. Negative controls (mouse or rabbit IgG) are also included. Scale bar = 5 μm. (B) Immunoblot of 231-mCherry breast cancer cells stably expressing vector or αB-crystallin. (C) Adhesion of 231-mCherry cells stably expressing vector or αB-crystallin (αB) to HBMECs. Cells were seeded onto confluent HBMECs for 2 h, washed and attached mCherry-positive cells were scored (mean ± SEM, n = 3, **P < 0.01). (D) Transendothelial migration (TEM) of 231-mCherry-Vector or 231-mCherry-αB cells at 24 h (mean ± SEM, n = 3, **P < 0.01). (E) BBB transmigration in vitro. 231-mCherry-Vector or 231-mCherry-αB cells that transmigrated through two cell layers (HBMECs and astrocytes) were scored at 48 h (mean ± SEM, n = 3, *P < 0.05).
Figure 3
Figure 3. Silencing αB-crystallin in TNBC cells inhibits adhesion to brain endothelium, transendothelial migration, and BBB transmigration in vitro
(A) Immunoblot of GILM2-mCherry breast cancer cells stably expressing a non-silencing (NS) construct, shRNAs targeting different αB-crystallin sequences (sh1-αB and sh2-αB), or sh1-αB and an αB-crystallin mutant that disrupts gene silencing but does not alter its coding sequence (sh1-αBM). (B) Adhesion of GILM2-mCherry cells stably expressing NS, sh1-αB, sh2-αB or sh1-αBM to HBMECs. Cells were seeded onto confluent HBMECs for 4 h, washed and attached mCherry-positive cells were scored (mean ± SEM, n = 3). (C) Transendothelial migration (TEM) of GILM2-mCherry cells stably expressing NS, sh1-αB, sh2-αB or sh1-αBM at 48 h (mean ± SEM, n = 3). (D) BBB transmigration in vitro of GILM2-mCherry cells stably expressing NS, sh1-αB, sh2-αB or sh1-αBM at 72 h (mean ± SEM, n = 3). For (B) – (D), *P < 0.05, **P < 0.01, ***P < 0.001 versus NS.
Figure 4
Figure 4. αB-crystallin promotes adhesion to HBMECs at least in part by an α3β1 integrin-dependent mechanism and enhances adhesion to extracellular matrix proteins
(A) The cell surface expression of various integrins in 231-mCherry breast cancer cells stably expressing vector or αB-crystallin (upper panels) and GILM2-mCherry cells stably expressing NS or sh-αB (lower panels) was determined using an α/β Integrin-mediated Cell Adhesion Array. Data is expressed as absorbance at 570 nm (mean ± SEM, n = 3). A negative control (c) was included. (B) 231-mCherry-Vector, 231-mCherry-αB, GILM2-mCherry-NS, and GILM2-sh-αB cells were preincubated with integrin blocking Abs for 1 h, seeded onto confluent HBMECs for 2h (231-mCherry cells) or 4h (GILM2-mCherry cells), washed, and attached mCherry-positive cells were scored (mean ± SEM, n = 3). (C) Adhesion of breast cancer cells to different ECM proteins. Cells were plated on fibronectin, collagen or laminin coated 24-well plates for 20 min (231-mCherry cells) or 30 min (GILM2-mCherry cells) at 37°C, washed, and attached cells were counted (mean ± SEM, n = 3). For (A) – (C), *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. αB-crystallin overexpression increases brain metastases in an orthotopic TNBC model
231-mCherry cells stably expressing vector or αB-crystallin (αB) were injected bilaterally into the ducts of the 4th mammary gland of NSG mice. (A) Mammary tumor volume expressed as the percentage original tumor volume at 2 weeks in mice with 231-mCherry-Vector and 231-mCherry-αB xenografts (n=10 mice per group). (B) Immunoblot of 231-mCherry-Vector and 231-mCherry-αB mammary tumors and αB-crystallin IHC staining of mammary tumors and brain metastases in both groups. (C) Representative fluorescent whole brain images from mice with 231-mCherry xenografts overexpressing vector or αB. (D) Number of mCherry-fluorescent metastases per brain in vector and αB groups, and percentage surface area of brain metastases in vector and αB groups (mean ± SEM, n = 10 mice per group, ***P < 0.001).
Figure 6
Figure 6. Silencing αB-crystallin inhibits brain metastases in an orthotopic TNBC model
GILM2-mCherry cells stably expressing a non-silencing (NS) construct or αB-crystallin shRNA (sh1-αB) were injected bilaterally into the ducts of the 4th mammary gland of immunodeficient NSG mice. (A) Mammary tumor volume expressed as the percentage original tumor volume at 2 weeks in mice with GILM2-mCherry-sh-αB and GILM2-mCherry-NS xenografts (n=10 mice per group). (B) Immunoblot of GILM2-mCherry-NS and GILM2-sh-αB mammary tumors and αB-crystallin IHC staining of mammary tumors and brain metastases in both groups. (C) Representative fluorescent whole brain images and number of fluorescent metastases per brain for NS and sh-αB groups (mean ± SEM, n = 10 mice per group, *P < 0.05). (D) Representative H&E staining of brain sections and number of metastatic lesions per section for NS and sh-αB groups (mean ± SEM, ***P < 0.001).
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