IntroductionThe irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs.MethodsTwo metastatic human breast cancer cell lines (MDA-MB 231 and BCM2) were used to optimize the conditions of hypoxia and reoxygenation cycles. The percentage of CSCs in the cycling hypoxia selected subpopulation was analyzed based on the CD44, CD24, ESA, and E-cadherin expression by three-color flow cytometry. Colony formation assays were used to assess the ability of this subpopulation to self-renew. Limiting dilution assays were performed to evaluate the tumor-initiating and metastatic ability of this subpopulation. Induction of EMT was examined by the expression of EMT-associated markers and EMT-associated microRNAs.ResultsUsing an optimized hypoxia and reoxygenation regimen, we identified a novel cycling hypoxia-selected subpopulation from human breast cancer cell lines and demonstrated that a stem-like breast cancer cell subpopulation could be expanded through repetitive hypoxia/reoxygenation cycles without genetic manipulation. We also found that cells derived from this novel subpopulation form colonies readily, are highly tumorigenic in immune-deficient mice, and exhibit both stem-like and EMT phenotypes.ConclusionsThese results provide the validity to the newly developed hypoxia/reoxygenation culture system for examining the regulation of CSCs in breast cancer cell lines by niche factors in the tumor microenvironment and developing differential targeting strategies to eradicate breast CSCs.

Metastasis, which remains incompletely characterized at the molecular and biochemical levels, is a highly specific process. Despite the ability of disseminated cancer cells to intravasate into distant tissues, it has been long recognized that only a limited subset of target organs develop clinically overt metastases. Therefore, subsequent adaptation of disseminated cancer cells to foreign tissue microenvironment determines the metastatic latency and tissue tropism of these cells. As a result, studying interactions between the disseminated cancer cells and the adjacent stromal cells will provide a better understanding of what constitutes a favorable or unfavorable microenvironment for disseminated cancer cells in a tissue-specific manner. Previously, we reported a protein signature of brain metastasis showing increased ability of brain metastatic breast cancer cells to counteract oxidative stress. In this study, we showed that another protein from the brain metastatic protein signature, neurotrophin-3 (NT-3), has a dual function of regulating the metastatic growth of metastatic breast cancer cells and reducing the activation of immune response in the brain. More importantly, increased NT-3 secretion in metastatic breast cancer cells results in a reversion of mesenchymal-like (EMT) state to epithelial-like (MET) state and vice versa. Ectopic expression of NT-3 in EMT-like breast cancer cells reduces their migratory ability and increases the expression of HER2 (human epidermal growth factor receptor 2) and E-cadherin at the cell–cell junction. In addition, both endogenous and ectopic expression of NT-3 reduced the number of fully activated cytotoxic microglia. In summary, NT-3 appears to promote growth of metastatic breast cancer cells in the brain by facilitating the re-epithelialization of metastatic breast cancer cells and downmodulating the cytotoxic response of microglia. Most importantly, our results provide new insights into the latency and development of central nervous system macrometastases in patients with HER2-positive breast tumors and provide mechanistic rationale to target HER2 signaling for HER2-positive breast cancer brain metastasis.

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.