Migration of metastatic BC cell lines to pre-OBs.

<p>(a) <i>Conditioned media (CM) of pre-OBs provide a migratory advantage to MDA-MB231 cells</i>. The wound healing assay was performed to assess migration of the MDA-MB231 BC cell line in the presence of CM from differentiating OBs derived from HD-BMSCs and KM105 cells. Results are expressed as percentage of negative control. Statistical analysis was performed with the ANOVA test. (b) and (c) <i>CM of pre-OBs support migration of metastatic BC cells</i>. Three different BC cell lines, two metastatic (MDA-MB231 and HCC-1954), and one invasive, but non-metastatic (MCF7); and one benign breast cell line (MCF10A), were subjected to the wound healing assay in the presence of CM from HD-BMSC- (b) or KM105-derived (c) pre-OBs or undifferentiated cells. Results are expressed as percentage of negative control. (d) <i>CM of pre-OBs do not affect BC cell survival</i>. MDA-MB231 cells were cultured in the presence of CM from KM105-derived osteolineage cells for three days. Cell survival was assessed with AlamarBlue assay.</p

MET inhibition overcomes HGF and pre-OB-induced migration of BC cells.

<p>(a) <i>MET inhibition by INCB28060 prevents HGF-induced MET phosphorylation in MDA-MB231 and HCC-1954 cells</i>. After one hour preincubation with INC2B8060, BC cells were stimulated with HGF for one hour. Phosphorylation of MET and total MET were determined by western blot and quantified by densitometric analysis. Results are expressed as percentage of unstimulated and untreated cells (negative control). <i>(d) MET inhibition by INC2B8060 or siMET prevent HGF-induced migration of BC cells</i>. 8-hour migration of MDA-MB231, HCC-1954 and MCF7 cells transfected with mock or siMET in the presence of HGF with or without INCB28060 was evaluated by a wound healing assay. (e) <i>MET inhibition impairs the migration advantage of MDA-MB231 BC cells provided by pre-OBs</i>, <i>but not undifferentiated cells</i>. After one hour preincubation with INCB28060, MDA-MB231 BC cells transfected with mock or siMET were exposed to CM of KM105-derived pre-OBs or undifferentiated cells and migration was assessed by wound healing assay. Percentage of negative control is shown. (f) <i>MET inhibition impairs the migration advantage of HCC-1954 BC cells provided by pre-OBs</i>. After one hour preincubation with INCB28060, BC cells transfected with mock or siMET were exposed to CM of KM105-derived pre-OBs in the presence of INCB28060 to assess migration by a wound healing assay. Percentage of negative control is shown.</p

Schematic representation of the interactions between pre-OBs and tumor cells.

<p>Pre-OBs enhance migration of breast cancer (BC) cells via activation of the HGF/MET pathway. In addition, metastatic BC cells preferentially adhere to pre-OBs.</p

Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells <i>via</i> the HGF/MET Pathway

<div><p>Introduction</p><p>The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown.</p><p>Methods and Results</p><p>Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALP^low OPN^low RUNX2^high OSX ^high CD166^high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, <i>HGF</i> mRNA was significantly up-regulated in pre-OBs <i>versus</i> mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration.</p><p>Conclusions</p><p>Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.</p></div