METHODS: A
versican G3 construct was exogenously expressed in the 66c14 and the MC3T3-E1 cell line. Cells were observed through light microscopy and viability analyzed by Coulter Counter or determined with colorimetric proliferation assays. The
Annexin V-FITC apoptosis detection kit was used to detect apoptotic activity. Modified Chemotactic Boyden chamber migration invasion assays were applied to observe
tumor migration and invasion to bone stromal cells and MC3T3-E1 cells.
Alkaline phosphatase (ALP) staining and ALP ELISA assays were performed to observe ALP activity in MC3T3-E1 cells.
RESULTS: In the four mouse
breast cancer cell lines 67NR, 66c14, 4T07, and 4T1, 4T1 cells expressed higher levels of
versican, and showed higher migration and invasion ability to MC3T3-E1 cells and primary bone stromal cells. 4T1
conditioned medium (CM) inhibited MC3T3-E1 cell growth, and even lead to apoptosis. Only 4T1 CM prevented MC3T3-E1 cell differentiation, noted by inhibition of
alkaline phosphatase (ALP) activity. We exogenously expressed a
versican G3 construct in a cell line that expresses low
versican levels (66c14), and observed that the G3-expressing 66c14 cells showed enhanced cell migration and invasion to bone stromal and MC3T3-E1 cells. This observation was prevented by selective EGFR inhibitor
AG1478, selective
MEK inhibitor
PD 98059, and selective AKT inhibitor
Triciribine, but not by selective JNK inhibitor SP 600125.
Versican G3 enhanced
breast cancer cell invasion to bone stromal cells or osteoblast cells appears to occur through enhancing EGFR/ERK or AKT signaling. G3 expressing MC3T3-E1 cells showed inhibited cell growth and cell differentiation when cultured with TGF-β1 (1 ng/ml), and expressed enhanced cell apoptosis when cultured with TNF-α (2 ng/ml). Enhanced EGFR/JNK signaling appears to be responsible for G3 enhanced osteoblast apoptosis and inhibited osteoblast differentiation. Whereas repressed expression of GSK-3β (S9P) contributes to G3 inhibited osteoblast growth.
Versican G3 functionality was dependent on its
EGF-like motifs. Without the structure of
EGF-like repeats, the G3 domain would not confer enhancement of
tumor cell migration and invasion to bone with concordant inhibition of osteoblast differentiation and promotion of osteoblast apoptosis.
CONCLUSIONS:
Versican enhances
breast cancer bone
metastasis not only through enhancing
tumor cell mobility, invasion, and survival in bone tissues, but also by inhibiting pre-osteoblast cell growth, differentiation, which supply favorable microenvironments for
tumor metastasis.