Abstract A1: NT-3 modulates HER2 signaling to promote the metastatic growth of HER2-positive breast cancer brain metastasis

Abstract

Abstract The purpose of our study is to determine the role of neurotrohpin-3 in promoting the growth of HER2-positive breast cancer brain metastasis in vivo. The establishment of metastatic-permissive microenvironment may require adaptation of metastatic cancer cells and hijacking of cellular immune responses. To understand the complexity of breast cancer metastasis in hopes of identifying targets and developing drugs against brain metastasis, we utilized large-scale mass-spectrometry-based proteomics to identify differentially expressed proteins in metastatic breast cancer cells with propensity to colonize the lung, the bone, and brain. We detected 250 proteins that were specifically up- or downregulated in the brain-colonizing breast cancer cells and found that a growth factor, neurotrophin-3 (NT-3) was specifically upregulated in brain colonizing breast cancer cells. The expression of NT-3 was particularly high in the HER2 positive brain colonizing breast cancer cell line, MDA-MB 361. Since brain metastases often appears in patients long after the resection of primary breast tumors (5–15 years) and NT-3 expression is low in primary breast tumors, we believe that adaptation to the brain microenvironment is the rate limiting factor for the development of breast cancer brain metastasis and examined the hypothesis that NT-3 expression promotes the formation of macrometastasis from metastatic breast cancer cells in the brain. We showed that by implanting metastatic breast cancer intracranially at low cell numbers, brain parenchyma permits the growth of brain colonizing metastatic breast cancer cell lines such as MDA-MB 361 cells and suppresses of unselected metastatic breast cancer cell lines such as MDA-MB 231 cells. The metastatic growth of GFP-expressing breast cancer cells in vivo can be measured quantitatively by the scanning confocal microscope. We discovered a novel NT-3 induced HER2 signaling in the development of HER2-positive breast cancer brain metastasis. Using a brain colonizing breast cancer cell line, MDA-MB 361, with endogenously high level of NT-3 and HER2, we found NT-3 signals through HER2 by inducing HER2 phosphorylation and NT-3/HER2 signaling can be inhibited by small molecule inhibitors targeting HER2 activation. More importantly, NT-3/HER2 signaling promotes the luminal epithelial phenotype and suppresses the epithelialmesenchymal transition (EMT)-like phenotype in metastatic breast cancer cells. By knocking down NT-3 in MDA-MB 361 cells, we found NT-3 expression is necessary to sustain the metastatic growth of brain colonizing breast cancer cells in the brain. Knocking down NT-3 reduced the expression of HER2 and E-cadherin and increased the expression of EMT-inducing transcription factor, Snail, in the nucleus. By overexpressing NT-3 in breast cancer cells with a poor brain-colonizing ability such as MDA-MB 231, we found NT-3 expression is sufficient to promote breast cancer brain metastasis. Ectopic expression of NT-3 increased HER2 and E-cadherin at the cell-cell junction and suppressed the nuclear expression of Snail and migratory ability of MDA-MB 231 breast cancer cells. Furthermore, both endogenous and ectopic expression of NT-3 reduced the number of fully activated cytotoxic microglia. In conclusion, our data suggest that NT-3 promotes EMT to MET conversion of metastatic breast cancer cells and allows brain colonizing cells to evade the cytotoxic immune response from the resident CNS immune cells, microglia. Most importantly, our results provide new insights into the latency and development of CNS macrometastases in patients with HER2-positive breast tumors and provide mechanistic rationale to target HER2 signaling for HER2-positive breast cancer brain metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A1.