Effect of heterogeneity of HER2 expression on brain metastases of breast cancer.

Abstract

635 Background: HER2-overexpressing or triple-negative [ER(-)/PR(-)/HER2(-)] breast cancers are associated with increased risk of brain metastases. The mechanisms leading to metastasis in each subtype are not well known. Methods: We introduced the wild-type HER2 gene into MDA-MB-231-luc-D3H2LN (231-Luc) cells, which are triple-negative breast cancer cells, and established HER2-expressing (63.2%) cells as 231-Luc-HER2 cells. We investigated the tumor formation following orthotopic inoculation and brain metastasis following intracardiac injection into nude mice. Metastasis was detected by bioluminescence imaging and confirmed in H&E staining and immunohistochemistry of vimentin and HER2 expressions. Flow cytometry analysis was used to detect the proportion of CD44+/CD24- cells, a maker for stem-like breast cancer cells. Results: 231-Luc-HER2 cells formed larger tumors in orthotopic xenograft models compared to 231-Luc cells, however, no significant difference was observed in proliferation in vitro. Neither 231-Luc-HER2 nor 231-Luc metastasized in the brain from the breast after orthotopic inoculation. After intracardiac injections of the 231-Luc-HER2 cells, brain metastasis developed (7/13 mice, 53.8%). Immunohistochemical analysis revealed that most metastasized cells expressed HER2, although we had injected a mixture of HER2-positive and HER2-negative cancer cells. Interestingly, administering Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, effectively prevented HER2-positive cells to colonize the brain. However, the HER2-negative 231-Luc-HER2 cells developed into brain metastases. In fact, the 231-Luc cells, which are HER2-negative, also metastasized in the brain (10/16 mice, 62.5%). Flow cytometry analysis of the 231-Luc-HER2 cells showed that HER2-positive cells decreased the population of CD44+/CD24- (HER2+/CD44+/CD24-: 86.8% and HER2-/CD44+/CD24-: 96.3%). Conclusions: The mechanism of brain metastases of HER2-positive breast cancer cells is different from that of HER2-negative breast cancer cells. It is therefore important to consider an additional therapeutic approach when dealing with HER2-negative cells in tumors having the heterogeneity of HER2 expression.