A 45-kDa ErbB3 secreted by prostate cancer cells promotes bone formation

Abstract

ErbB3 is a transmembrane growth factor receptor that has been implicated in the pathogenesis of human cancer. After finding that a truncated form of ErbB3 was present and upregulated in metastatic prostate cancer cells in lymph nodes and bone, we explored the pathophysiological functions of this unusual form of ErbB3 in the context of mouse calvaria as well as osteoblasts in vitro and the femur microenvironment in vivo. Here we demonstrate that prostate cancer cells expressed an alternatively spliced transcript that encodes a 45-kDa glycosylated protein (p45-sErbB3). The recombinant p45-sErbB3 purified from conditioned medium stimulated calvarial bone formation and induced osteoblast differentiation. Overexpression of p45-sErbB3 in the osteolytic prostate cancer cell line PC-3 converted its phenotype from bone lysing to bone forming upon injection into the femurs of immunodeficient mice. Further, we detected sErbB3 in plasma samples from patients with castration-resistant prostate cancer with bone metastasis. These observations establish that p45-sErbB3 is a structurally and functionally unique gene product of ErbB3 and suggest that p45-sErbB3 is likely one of the factors involved in the osteoblastic bone metastases of prostate cancer.

Figure 1

Prostate cancer cells express an alternative ErbB3 transcript encoding for a secreted protein. (A) Structures of p45-sErbB3 and p180-ErbB3. p180-ErbB3 contains four ligand-binding subdomains (I through IV), a single transmembrane (TM) domain, and a cytoplasmic domain. Two alternatively-spliced sequences from GenBank may encode p45-sErbB3. One transcript (BC002706 or BT007226) has exon 8 to exon 26 splicing and the other (U88358) has intron 8 retention. Translation of the latter transcript is expected to terminate after exon 8 due to a shift in the open reading frame. (B) Prostate cancer cells produce an alternatively-spliced ErbB3 transcript. RT-PCR of the RNA from the three prostate cancer cell lines, using primers specific to the exon 6–exon7 junction and to intron 8, generated a 326-bp transcript—the predicted size from U88358 (left). This amplicon hybridized with an oligonucleotide probe specific to exon 8 (right), confirming that it is an alternatively-spliced ErbB3 transcript. (C) The alternatively spliced transcript encodes a secreted protein. LNCaP, PC-3, and DU145 prostate cancer cells were infected with Ad-p45 (p45) or with Ad-luciferase (C) for 48 h, after which the conditioned medium (CM) and cell lysates (Lys) were analyzed for the expression of p45-sErbB3 by western blotting. (D) p45-sErbB3 is a glycoprotein. CM from Ad-p45 infected cells was treated (+) or not treated (−) with peptidyl N-glycosidase F (PNGase) for 1 h and analyzed by western blotting.

Figure 2

p45-sErbB3 stimulates bone formation and osteoblast activity in vitro. (A) Recombinant p45-sErbB3 from baculovirus-infected Sf9 cells was purified by metal affinity chromatography. Recombinant p45-sErbB3 stimulates new bone formation in a mouse calvarial culture assay. Neonatal mouse calvaria was treated (+) or not treated (−) with the indicated amounts of recombinant p45-sErbB3 for 4 days. Histologic analysis of the calvaria showed increased thickness in the calvaria that had been treated with p45-sErbB3 but not in the corresponding controls. Increases in osteoblast numbers, total bone area, and new bone area in the calvaria treated with p45-sErbB3 (100 ng/ml) relative to those in controls. Error bars represent standard deviation; *significant difference at P <0.001. (B) Effect of recombinant p45-sErbB3 from Sf9 cells on osteoblast proliferation. Primary mouse osteoblasts (PMOs) isolated from neonatal mouse calvaria were treated or not treated with various concentrations of purified p45-sErbB3 protein. *Significant difference at P <0.05. (C) Effect of recombinant p45-sErbB3 from mammalian cells on osteoblast proliferation. Recombinant p45-sErbB3, purified from p45-sErbB3 adenovirus-infected PC-3 cells, or recombinant p45-sErbB3 adenovirus (Ad-p45) at various multiplicities of infection (MOI), was used to treat PMOs isolated from neonatal mouse calvaria. Ad-luciferase was used as a control. *Significant difference at P <0.05. (D) Alkaline phosphatase activity in PMO cultures not treated (control) or treated with p45-sErbB3 (100 ng/ml) in differentiation medium for 15 days. Results are expressed as means; error bars are standard deviation; *significant difference at P <0.05. (E) Formation of bone-like nodules in PMO cultures that were either not treated (control) or treated with p45-sErbB3 (100 ng/ml) for 15 days in differentiation medium, followed by von Kossa staining.

Figure 3

p45-sErbB3 does not stimulate PC-3 cell proliferation in vitro or in vivo. (A) Conditioned medium from PC/neo and PC/p45-sErbB3 cells. Expression of FLAG-tagged p45-sErbB3 was confirmed by immunoprecipitation with anti-FLAG-agarose and western blotting with the anti-ErbB3 antibody Ab-10. (B) p45-sErbB3 does not stimulate PC-3 cell proliferation in vitro. (C) p45-sErbB3 does not increase PC-3 cell growth at subcutaneous sites in vivo in nu/nu mice, in which PC/neo or PC/p45-sErbB3 cells were injected subcutaneously into the flanks of nu/nu mice (1 × 10⁶ cells/site × 12 sites) and the resulting tumors were measured weekly thereafter. Values shown are means; bars represent standard error.

Figure 4

p45-sErbB3 induces a bone-forming phenotype in the mouse femurs. (A) PC/neo or PC/p45-sErbB3 cells were injected into the distal ends of the right femurs of 12-week-old male CB.17 SCID mice; left femurs were injected with saline as a control. Severe osteolytic effects (circle) were seen in mouse femurs at 4 weeks after injection of PC/neo cells. Histologic analysis of lesions induced by intrafemoral injection of PC/neo cells at 6 weeks showed a large tumor (T) with severe bone lysis (x40) and osteoclasts (arrows) at the area invaded by tumor (x100). However, no bone lysis (circle) was seen in femurs injected with PC/p45-sErbB3 cells. Histologic analysis of lesions induced by PC/p45-sErbB3 cells revealed the appearance of new bone surrounding the tumor (T) (x40) and activated osteoblasts (arrowheads) within the woven bone (x100). (B) Histologic analysis of femur lesions induced by the selective PC/p45-sErbB3 clonal cell lines. The newly formed bone (B*) in the tumor (T) –invaded area has features of “woven bone” that contrast with the appearance of normal bone (B), in which collagen fibers are arranged in lamellae. The dark-staining cells are normal bone marrow cells.

Figure 5. Levels of sErbB3 in the conditioned medium of prostate cancer cell lines and human plasma

(A) Conditioned medium from LNCaP, PC-3, or DU145 cells grown to confluence on 10-cm tissue culture plates was concentrated 20-fold using a Centricon concentrator. ELISA was performed using 100 μL each of the concentrated conditioned medium. The data were expressed as ng/mL of original conditioned medium. (B) Conditioned medium from PC/neo and PC/p45-sErbB3 clones #16, #18, #19 was concentrated 20-fold and the concentration of sErbB3 measured by ELISA. The data were expressed as ng/mL of original conditioned medium. (C) The concentrations of sErbB3 in the plasma samples from 50 men with progressive castration-resistant prostate cancer with bone metastasis were measured by ELISA. The samples are ordered from the lowest to highest sErbB3 levels from left to right on the x axis. a-f, samples with sErbB3 concentrations higher than 3 ng/mL sErbB3 and they are 3.9 ± 0.3, 5.1 ± 0.2, 7.5 ± 1.2, 9.1 ± 3.0, 11.7 ± 2.5, and 11.8 ± 2.7, for a-f, respectively.