A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of β-catenin remained uninfluenced. Consistently, β-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. β-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

ABSTRACT:The bisphosphonate zoledronic acid (ZA) significantly reduces complications of bone metastasis by inhibiting resident macrophages, the osteoclasts. Recent clinical trials indicate additional anti-metastatic effects of ZA outside the bone. However, which step of metastasis is influenced and whether this is due to direct toxicity on cancer cells or inhibition of the tumor promoting microenvironment, is unknown. In particular, tumor-associated and resident macrophages support each step of organ metastasis and could be a crucial target of ZA.Thus, we comparatively investigate the ZA effects on: i) different types of macrophages, ii) on breast cancer cells but also iii) on macrophage-induced invasion. We demonstrate that ZA concentrations reflecting the plasma level affected viability of human macrophages, murine bone marrow-derived macrophages as well as their resident brain equivalents, the microglia, while it did not influence the tested cancer cells. However, the effects on the macrophages subsequently reduced the macrophage/microglia-induced invasiveness of the cancer cells. In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells. The characterization of human macrophages after ZA treatment revealed a phenotype/response shift, in particular after external stimulation.In conclusion, we show that therapeutic concentrations of ZA affect all types of macrophages but not the cancer cells. Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment. Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis.

Liver metastasis development in breast cancer patients is common and confers a poor prognosis. So far, the prognostic significance of surgical resection and clinical relevance of biomarker analysis in metastatic tissue have barely been investigated. We previously demonstrated an impact of WNT signaling in breast cancer brain metastasis. This study aimed to investigate the value of established prognostic markers and WNT signaling components in liver metastases. Overall N = 34 breast cancer liver metastases (with matched primaries in 19/34 cases) were included in this retrospective study. Primaries and metastatic samples were analyzed for their expression of the estrogen (ER) and progesterone receptor, HER-2, Ki67, and various WNT signaling-components by immunohistochemistry. Furthermore, β-catenin-dependent and -independent WNT scores were generated and analyzed for their prognostic value. Additionally, the influence of the alternative WNT receptor ROR on signaling and invasiveness was analyzed in vitro. ER positivity (HR 0.09, 95 % CI 0.01–0.56) and high Ki67 (HR 3.68, 95 % CI 1.12–12.06) in the primaries had prognostic impact. However, only Ki67 remained prognostic in the metastatic tissue (HR 2.46, 95 % CI 1.11–5.44). Additionally, the β-catenin-independent WNT score correlated with reduced overall survival only in the metastasized situation (HR 2.19, 95 % CI 1.02–4.69, p = 0.0391). This is in line with the in vitro results of the alternative WNT receptors ROR1 and ROR2, which foster invasion. In breast cancer, the value of prognostic markers established in primary tumors cannot directly be translated to metastases. Our results revealed β-catenin-independent WNT signaling to be associated with poor prognosis in patients with breast cancer liver metastasis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-016-9780-3) contains supplementary material, which is available to authorized users.

The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown.Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression.Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis.

An essential function of the transcription factors LEF1/TCF4 in cerebral metastases of lung adenocarcinomas has been described in mouse models, suggesting a WNT/β-catenin effect as potential mechanism. Their role in humans is still unclear, thus we analyzed LEF1, TCF4, β-catenin, and early stage prognostic markers in 25 adenocarcinoma brain metastases using immunohistochemistry (IHC). IHC revealed nuclear TCF4 in all adenocarcinoma samples, whereas only 36 % depicted nuclear LEF1 and nuclear β-catenin signals. Samples with nuclear LEF1 as well as high TCF4 (++++) expression were associated with a shorter survival (p = 0.01, HR = 6.68), while nuclear β-catenin had no significant impact on prognosis and did not significantly correlate with nuclear LEF1. High proliferation index Ki67 was associated with shorter survival in late-stage disease (p = 0.03, HR 3.27). Additionally, we generated a LEF1/TCF4 as well as an AXIN2 signature, the latter as representative of WNT/β-catenin activity, following a bioinformatics approach with a gene expression dataset of cerebral metastases in lung adenocarcinoma. To analyze the prognostic relevance in primary lung adenocarcinomas, we applied both signatures to a microarray dataset of 58 primary lung adenocarcinomas. Only the LEF1/TCF4 signature was able to separate clusters with impact on survival (p = 0.01, HR = 0.32). These clusters displayed diverging enrichment patterns of the cell cycle pathway. In conclusion, our data show that LEF1/TCF4, but not β-catenin, have prognostic relevance in primary and cerebrally metastasized human lung adenocarcinomas. In contrast to the previous in vivo findings, these results indicate that LEF1/TCF4 act independently of β-catenin in this setting.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-012-9552-7) contains supplementary material, which is available to authorized users.