Loss of p16/Ink4a drives high frequency of rhabdomyosarcoma in a rat model of Duchenne muscular dystrophy

Abstract

Rhabdomyosarcoma (RMS) is an aggressive type of soft tissue sarcoma, and pleomorphic RMS is a rare subtype of RMS found in adult. p16 is a tumor suppressor which inhibits cell cycle. In human RMS, p16 gene is frequently deleted, but p16-null mice do not develop RMS. We reported that genetic ablation of p16 by the crossbreeding of p16 knock-out rats (p16-KO rats) improved the dystrophic phenotype of a rat model of Duchenne muscular dystrophy (Dmd-KO rats). However, p16/Dmd double knock-out rats (dKO rats) unexpectedly developed sarcoma. In the present study, we raised p16-KO, Dmd-KO, and dKO rats until 11 months of age. Twelve out of 22 dKO rats developed pleomorphic RMS after 9 months of age, while none of p16-KO rats and Dmd-KO rats developed tumor. The neoplasms were connected to skeletal muscle tissue with indistinct borders and characterized by diffuse proliferation of pleomorphic cells which had eosinophilic cytoplasm and atypical nuclei with anisokaryosis. For almost all cases, the tumor cells immunohistochemically expressed myogenic markers including desmin, MyoD, and myogenin. The single cell cloning from tumor primary cells gained 20 individual Pax7-negative MyoD-positive RMS cell clones. Our results demonstrated that double knock-out of p16 and dystrophin in rats leads to the development of pleomorphic RMS, providing an animal model that may be useful to study the developmental mechanism of pleomorphic RMS.

Keywords: MyoD; desmin; muscular dystrophy; rhabdomyosarcoma; skeletal muscle.

Fig. 1.

Gross and histopathological appearance of rhabdomyosarcoma in double knock-out rats. (A) The subcutaneous tumors (arrows) are located in the cheek, neck, peritoneal cavity and forelimb region. All the tumors are connected to skeletal muscles. (B) Tumor cells invade the adjacent muscle tissue and are now diffuse. Hematoxylin and eosin stain. Bar=250 μm. (C) Tumor cells showing indistinct cytoplasmic borders are arranged in an irregular bundle pattern. Hematoxylin and eosin stain. Bar=100 μm. (D) Sheets of pleomorphic round-to-ovoid cells are observed. Multinucleated giant cells are occasionally present (arrowhead). Hematoxylin and eosin stain. Bar=20 μm. (E) Sheets of pleomorphic spindle cells are observed. Aberrant mitotic figures are occasionally present (arrowhead). Hematoxylin and eosin stain. Bar=20 μm. (F) Striations are rarely present (arrowhead). Phosphotungstic acid hematoxylin stain. Bar=20 µm.

Fig. 2.

Immunohistochemical characteristics of rhabdomyosarcoma in double knock-out rats. (A) The tumor cells show focal cytoplasmic immunolabeling with desmin. The nuclei of the tumor cells weakly to moderately label with MyoD and myogenin. Arrowheads indicate immune-positive cells. Bar=50 μm. (B) The tumor cells in case 10 show focal cytoplasmic immunolabeling with desmin. The nuclei of the tumor cells weakly to moderately label with myogenin. The tumor cells are negative for MyoD. Bar=50 μm.

Fig. 3.

Immunocytochemical characteristics of rhabdomyosarcoma-derived primary cells from case 1. (A) The cells express strongly desmin, but moderately Pax7 and MyoD. The cells are negative for myogenin. Yellow arrowheads indicate immune-positive cells, and white arrowheads indicate immune-negative cells. Scale bar=250 μm. (B) Quantification of desmin, Pax7, MyoD, and myogenin-positive cells relative to total cell count. Data are expressed as means of 5 middle-power fields ± standard error.

Fig. 4.

Immunocytochemical characteristics of rhabdomyosarcoma-derived cloned cell lines from case 1. The cells express strongly desmin and MyoD. The cells are negative for Pax7 and myogenin. Scale bar=100 μm.