A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells

Abstract

The propensity for prostate cancer to metastasize to bone led us and others to propose that bidirectional interactions between prostate cancer cells and bone are critical for the preferential metastasis of prostate cancer to bone. We identified previously a secreted isoform of ErbB3 (p45-sErbB3) in bone marrow supernatant samples from men with prostate cancer and bone metastasis and showed by immunohistochemical analysis of human tissue specimens that p45-sErbB3 was highly expressed in metastatic prostate cancer cells in bone. Here, we show that p45-sErbB3 stimulated mouse calvaria to secrete factors that increased the invasiveness of prostate cancer cells in a Boyden chamber invasion assay. Using gene array analysis to identify p45-sErbB3-responsive genes, we found that p45-sErbB3 up-regulated the expression of osteonectin/SPARC, biglycan, and type I collagen in calvaria. We further show that recombinant osteonectin increased the invasiveness of PC-3 cells, whereas osteonectin-neutralizing antibodies blocked this p45-sErbB3-induced invasiveness. These results indicate that p45-sErbB3 enhances the invasiveness of PC-3 cells in part by stimulating the secretion of osteonectin by bone. Thus, p45-sErbB3 may mediate the bidirectional interactions between prostate cancer cells and bone via osteonectin.

Fig. 1

Factors secreted by osteoblasts exposed to p45-sErbB3 enhance the invasiveness of prostate cancer cells. A, experimental plan for examining the effect of p45-sErbB3 on mouse calvaria, both in organ-culture assays and in conditioned medium. B, histologic analysis indicates that treatment of calvaria with p45-sErbB3 increased calvarial bone formation. C, in transwell Boyden chambers, conditioned medium from p45-sErbB3–treated calvaria increased the invasiveness of PC-3 cells. Representative microscopic photographs show reassembled images of the entire transwell membrane, on which cells that migrated to the outside of the upper chamber were stained and counted. D, quantification of the invasiveness of PC-3, LNCaP, and C4-2B cells in response to conditioned medium from p45-sErbB3–treated calvaria in a transwell assay. *P < 0.05; **P < 0.01.

Fig. 2

p45-sErbB3 stimulates expression of osteonectin and other osteoblast genes in mouse calvaria. A, analysis of an osteogenic gene array showed the increased expression of osteonectin (Osn), biglycan (Bgn), and type I collagen (Col-1) in response to p45-sErbB3 exposure. GAPDH, glyceraldehyde-3-phosphate dehydrogenase (a control gene). B, northern blots of osteonectin, biglycan, and type I collagen mRNAs from calvaria treated or not treated with p45-sErbB3. 18s rRNA was used as internal control for calibrating the RNA loading. The experiment was repeated three times, with similar data obtained each time. “Fold” changes in osteonectin, biglycan, and type I collagen production induced by p45-sErbB3 are shown at right. *P < 0.05; ** P < 0.01. C, western blotting of osteonectin in conditioned medium from p45-sErbB3–treated calvaria. Bovine serum albumin was stained to show equal aliquots of media were loaded. The result was representative of three similar experiments. Mean differences in protein density from 3 experiments are shown at right. *P <0.05.

Fig. 3

Osteonectin mediates the effect of p45-sErbB3 on the invasiveness of prostate cancer cells. A, In Boyden transwell assays with the indicated osteonectin concentrations, cells that had migrated to the outside of the upper chamber after a 20-h incubation were counted. Osteonectin increased the invasiveness of PC-3 and C4-2B cells in a concentration-dependent manner. *P < 0.05; **P < 0.01. B, conditioned medium from calvaria treated or not treated with p45-sErbB3 was incubated with 1 μg/ml of anti-osteonectin rabbit polyclonal antibody (rPA), mouse monoclonal antibody (mAb), or control antibody at room temperature for 2 h and was subsequently added to the bottom wells of Boyden chambers for PC-3 cell invasion assay. Experiments were repeated three times, with similar data obtained each time; values shown are means of the triplicates. **P < 0.01. C, a diagram of putative p45-sErbB3–induced bidirectional interactions between prostate cancer cells and osteoblasts. The p45-sErbB3 has an osteoblastic effect on bone and stimulates the expression of osteonectin, which enhances prostate cancer cell invasiveness.