Constitutive Akt activation drives WDLPS in the zebrafish. (A) Experimental design. (B) Solid tumor incidence in p53 wild-type, heterozygous, or p53^M214K homozygous mutant siblings injected with a rag2:myr-mAkt2 transgene at the one-cell stage. P value calculated via log-rank test. (C) Representative rag2:myr-mAkt2-injected zebrafish, which developed what appear to be two independent solid tumors. The animal shown was p53-homozygous mutant. (Scale bar, 5 mm.) Note that the image shown in (C) consists of merged adjacent photomicrographs. (D) Control H&E-stained zebrafish section. Arrow points to normal subcutaneous adipocytes. (E) Low-magnification view of an H&E section through the zebrafish shown in C, demonstrating a locally invasive mass consisting of well-differentiated adipocytes with significant variation in cell size. (F) High-magnification view of H&E section demonstrating a representative lipoblast scattered throughout these tumors, characterized by a multivacuolated cytoplasm and large hyperchromatic pleomorphic nuclei. (G) Phospho-AKT immunohistochemistry on a control zebrafish section. Arrow points to normal subcutaneous adipocytes, which lacked detectable pAKT staining. (H and I) Phospho-AKT immunohistochemistry on tumor sections from the zebrafish shown in C, revealing strong immunoreactivity for phospho-AKT in tumor cells of rag2:myr-mAkt2-transgenic zebrafish. (Scale bars, 100 μm.)

Osteosarcoma development in a rag2:myr-mAkt2-injected, p53-homozygous mutant zebrafish. (A and B) A p53-homozygous mutant zebrafish injected with the rag2:myr-mAkt2 expression construct developed a solid lobulated mass at the base of the dorsal fin. Note that the image shown in A consists of merged adjacent photomicrographs. (Scale bars, 1 mm.) (C and D) H&E-stained sections at low and high magnification, respectively, demonstrate a mass consisting predominantly of osteoid matrix interspersed with large malignant cells with pleomorphic nuclei, features that in humans are diagnostic of osteoblastic osteosarcoma. (Scale bars, 100 μm.)

AKT pathway activation in primary human well-differentiated and dedifferentiated liposarcomas. (A–C) H&E staining of human lipoma, WDLPS, and DDLPS specimens. (D–F) Immunohistochemistry for Ser473-phosphorylated AKT in a representative lipoma and AKT-positive WDLPS and DDLPS clinical specimens. (Scale bar, 100 μm.) (G–I) Immunohistochemistry for phospho-S6 ribosomal protein (Ser235/236) in a representative lipoma, as well as AKT-positive WDLPS and DDLPS clinical specimens. (J and K) Quantitation of phospho-AKT and phospho-S6 immunohistochemistry in pure WDLPS, pure DDLPS, and in human tumors with mixed well-differentiated and dedifferentiated liposarcoma components.