Polyoma virus-induced osteosarcomas in inbred strains of mice: host determinants of metastasis

Abstract

The mouse polyoma virus induces a broad array of solid tumors in mice of many inbred strains. In most strains tumors grow rapidly but fail to metastasize. An exception has been found in the Czech-II/Ei mouse in which bone tumors metastasize regularly to the lung. These tumors resemble human osteosarcoma in their propensity for pulmonary metastasis. Cell lines established from these metastatic tumors have been compared with ones from non-metastatic osteosarcomas arising in C3H/BiDa mice. Osteopontin, a chemokine implicated in migration and metastasis, is known to be transcriptionally induced by the viral middle T antigen. Czech-II/Ei and C3H/BiDa tumor cells expressed middle T and secreted osteopontin at comparable levels as the major chemoattractant. The tumor cell lines migrated equally well in response to recombinant osteopontin as the sole attractant. An important difference emerged in assays for invasion in which tumor cells from Czech-II/Ei mice were able to invade across an extracellular matrix barrier while those from C3H/BiDa mice were unable to invade. Invasive behavior was linked to elevated levels of the metalloproteinase MMP-2 and of the transcription factor NFAT. Inhibition of either MMP-2 or NFAT inhibited invasion by Czech-II/Ei osteosarcoma cells. The metastatic phenotype is dominant in F1 mice. Osteosarcoma cell lines from F1 mice expressed intermediate levels of MMP-2 and NFAT and were invasive. Osteosarcomas in Czech-II/Ei mice retain functional p53. This virus-host model of metastasis differs from engineered models targeting p53 or pRb and provides a system for investigating the genetic and molecular basis of bone tumor metastasis in the absence of p53 loss.

Figure 1. Primary and metastatic bone tumors from CZ mice.

A) A primary bone tumor (P) on the head of the femur of a CZ mouse. B) H&E stained section of the same tumor showing invasion of adjacent muscle and destruction of bone by invading tumor cells. C) Metastatic nodule (Mx) on the surface of the lung of the same animal. D) H&E stained section of the lung metastasis from the same animal showing osteiod. E) Occult metastatic lesion with osteoid in the lung of a different CZ animal. F) H&E stained section of a liver metastasis showing osteoid in a CZ animal inoculated with osteosarcoma cells.

Figure 2. Osteopontin is the major chemoattractant inducing bone tumor cell migration.

A) Bone tumor cells (3×10⁵) were used in a Boyden chamber assay using conditioned medium from C3 bone tumor cells as chemoattractant. Vertical bars represent mean number of cells migrating ± S.D. Solid bars show migration using conditioned medium. Empty bars show migration using serum-free medium as a control. B) Tumor cell migration was assayed as in (A) using conditioned medium (CM) that was untreated (empty bars), pre-treated with anti-VEGF (hatched bars) or anti-OPN (solid bars). Left panel – CZ I cells with osteosarcoma CM; Middle panel – CZ I cells with hemangioma CM; Right panel – hemangioma cells with hemangioma CM. C) Tumor cell migration in response to recombinant OPN in serum-free medium (hatched bars) or serum-free medium alone (empty bars).

Figure 3. Metastatic tumor cells invade Matrigel via an MMP-2-mediated pathway.

A) Migration assay was carried out as in Figure 2A except the upper surface of the filter was coated with Matrigel. B) Upper – Zymography with gelatin as ‘in gel’ substrate and conditioned media from each of the tumor lines. Lower – Immunoblot for MMP-2 in tumor cell lysates.

Figure 4. Activation of NFAT and secretion of MMP-2 are critical for invasion by metastatic bone tumor cells.

A) Immunoblot for NFAT c1 in tumor cell lysates. B) Normalized luciferase activity of an NFAT reporter in tumor cells. C) Inhibition of invasion by CZ II tumor cells. Left: cells ± TIMP-2. Middle: cells ± NFAT siRNA. Right: immunoblots on cells ± NFAT siRNA.

Figure 5. Induction of p53 and p21^Cip1/Waf1 in a DNA damage response in metastatic and non-metastatic bone tumor cell lines.

Cell extracts were separated by SDS-PAGE and immunoblotted as indicated. ActD – actinomycin D.