BMP5 treatment of adrenocortical carcinoma delays tumor growth via activation of endogenous anti-tumoral macrophages

Bone morphogenetic proteins (BMPs) are known to affect a variety of biological processes and also have been reported to modulate tumor growth. Former investigations revealed loss or downregulation of BMP5 in adrenocortical cancer (ACC) samples compared to normal adrenal glands and a deficit of downstream signalling in the human ACC cell line NCI h295R. Nude mice were subcutaneously xenografted with NCI h295R cells to establish solid tumors and treated with intra-tumoral administration of human recombinant (hr) BMP5 protein. Upon BMP5 delivery we observed a significant reduction in tumor size in comparison to PBS treated animals and a reduced proliferation rate (Ki-67 index) was evident. The canonical SMAD1/5/8 signalling pathway was not relevant in total tumor tissue with no detectable changes in human ID1, ID3, SMAD 6/7 and SMURF gene expression and SMAD1/5/8 phosphorylation. As BMP6 is reported to activate macrophages towards a pro-inflammatory anti-tumoral phenotype we were interested in a possible activating effect of BMP5. Expression of murine IL-12p40 was upregulated and a slight increase in the anti-angiogenic chemokines Mig (CXCL-9), IP-10 (CXCL-10) and murine macrophage marker F4/80 was detectable in BMP5 treated total tumor tissue. We also detected an increase in human CCL-2 (MCP1) expression, a monocyte attractant protein. Immunohistochemistry for galectin-3 showed influx of inflammatory macrophage-like cells into tumor tissue following BMP5 delivery. Murine macrophage cells (RAW 264.7) were treated with hrBMP5 and an increase in murine TNF-α expression and in the expression of the differentiation-related macrophage transcription factors PU.1 and IRF-5 was detected as well as upregulation of murine ID1 and ID3. We assume that the BMP5 mediated reduction in tumor growth might be achieved not via intracellular BMP signalling but rather by activation of anti-tumoral macrophages exerting anti-angiogenic, cytostatic or cytotoxic function on ACC tumors.