Stable Spontaneous Fusion of Estrogen Receptor (ER) Positive and ER Negative Breast Cancer Cells Generates an Efficient Model for Breast Cancer Brain Metastasis.
Authors: K. De, A. Bandyopadhyay, A. Chang, A. Elkahloun, J. Cornell, J. Yang, L. Wang, I. Yeh, B. Goins, L. Sun
Journal: Cancer Research (2009)

Abstract Background: Nearly 20% of patients with advanced breast cancer are eventually diagnosed with brain lesions. Rodent model systems for brain metastasis induced by breast carcinoma have been reported but the current models have limitations as systemic treatments of breast cancer metastases are ineffective in brain metastasis due to their inability to penetrate the blood brain barrier. Previously we have characterized a cell line, B6TC, derived from the stable spontaneous fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow (SABCS-2009 abstract#2053). In the present study we have developed an efficient mouse model for studying breast cancer brain metastasis with B6TC to establish a refined molecular signature for brain metastasis.Methods and Results: To determine the brain metastatic potential of B6TC cells compared to MDA-MB-231 in vivo, both the cells were injected into the left cardiac ventricle of female nude mice. The number and size of brain metastases formed were analyzed by EGFP and PET/CT fusion imaging of live mice, which revealed that 30% of the mice in B6TC group developed metastases to brain after only 4 weeks, while none in the MDA-MB-231 group developed brain metastases. The brain was removed, fixed in formalin and paraffin-embedded sections were processed by H&amp;E staining for histology and immunostained for EGFP and human TP53. The presence of brain metastases was confirmed by detection of EGFP and TP53 in brain section. Most of the metastases in each section were classified as micrometastases, comprising few tumor cells. We performed RNA microarray to identify genes whose RNA levels are significantly altered in B6TC cells by greater than 2-fold at the false discovery rate of 0.001, when compared to the parental metastatic cell line MDA-MB-231 or non aggressive cell line ZR-75-1. From genome-wide expression analysis, genes implicated in brain metastasis regulation are shown in Table 1. The over or under expression of potent brain metastatic genes were also validated by Western Blot analysis and our data is consistent with earlier reports identifying some of these genes as potential breast cancer brain metastatic genes from patients.Conclusion: Our study indicates potential genes for breast cancer brain metastasis, which are upregulated (MMP1, HB-EGF, ST3GAL1, ITGA3, CXCR4, EREG, PTGS2) or downregulated (KISS1, BRMS1L). Over-expression or knocking down of these candidate genes in relevant cells to study the effect on brain metastasis is under way to unravel a refined molecular signature for breast cancer brain metastases in our novel model system, which will provide valuable insight into molecular mechanism of breast cancer brain metastasis.Table1: Genes differentially expressed among 3 cell lines in RNA Microarray (↑: upregulation; ↓: downregulation)GeneDescriptionExpressionMMP1Matrix metallopeptidase 1↑HB-EGFHeparin-binding EGF-like growth factor↑ST3GAL1ST3 beta-galactoside alpha-2,3-sialyltransferase 1↑ITGA3Integrin, alpha 3↑CXCR4Chemokine (C-X-C motif) receptor 4↑EREGEpiregulin↑PTGS2Prostaglandin-endoperoxide synthase 2↑KISS1KiSS-1 metastasis-suppressor↓BRMS1LBreast cancer metastasis-suppressor 1-like↓ Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6147.