Aberrant AKT activation drives well-differentiated liposarcoma

Abstract

Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its pathobiology and development of effective targeted therapies. Here, we show that zebrafish expressing constitutively active Akt2 in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53 wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53-homozygous mutant zebrafish (P = 0.013), indicating that aberrant Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of the closely related dedifferentiated liposarcoma (DDLPS) subtype, revealed immunohistochemical evidence of AKT activation in 27% of cases. Western blot analysis of a panel of cell lines derived from patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured in serum-free media. Moreover, BEZ235, a small molecule inhibitor of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar concentrations. Our findings are unique in providing an animal model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this chemoresistant sarcoma.L iposarcoma is the most common sarcoma of humans, affecting ∼2,000 individuals per year in the United States (1). These tumors are classified into five histopathologic subtypes, with well-differentiated liposarcoma (WDLPS) accounting for ∼50% of cases, and dedifferentiated liposarcoma (DDLPS), a closely related subtype that appears to arise from further malignant progression of WDLPS, accounting for an additional 9% to 18% of cases (1-3). Liposarcomas are generally thought to arise de novo rather than from preexisting benign lesions, and most patients lack recognized causative factors. Although complete surgical resection can be curative, WDLPS often develops in deep anatomic locations, such as the retroperitoneum or mediastinum, where its propensity to enwrap vital structures typically makes complete surgical resection difficult or impossible, leading to high morbidity and mortality rates (1, 4). Radiation and chemotherapy have limited efficacy in the treatment of WDLPS (5, 6). Indeed, there are no systemic therapeutic regimens known to improve survival when complete surgical resection is not feasible, underscoring the need for an improved molecular understanding of WDLPS to stimulate the development of effective targeted therapies.The MDM2-p53 pathway plays a prominent role in WDLPS pathogenesis, with the vast majority of human tumors harboring either MDM2 amplifications or p53 mutations (6-10). Moreover, individuals with germ-line p53 mutations appear to be at increased risk of WDLPS development at a very young age (11).Regions of chromosome 12q13...

Zebrafish Models for Human Cancer

“…61 In a similar approach, the zebrafish rag2 promoter driving myristoylated, constitutively active mouse Akt2 was used to develop a transgenic model for well-differentiated liposarcoma. 38 Although tumor incidence was low in a wild-type background, 29% of transgenic zebrafish with concomitant homozygous tp53 mutation developed liposarcomas. 38 Liposarcomas were composed of well-differentiated lipocytes intermixed with atypical stromal cells and lipoblasts with pleomorphic nuclei and were locally invasive.…”

“…38 Although tumor incidence was low in a wild-type background, 29% of transgenic zebrafish with concomitant homozygous tp53 mutation developed liposarcomas. 38 Liposarcomas were composed of well-differentiated lipocytes intermixed with atypical stromal cells and lipoblasts with pleomorphic nuclei and were locally invasive. 38 Although rhabdomyosarcoma and liposarcoma models were developed by transgenic approaches, a model for hemangiosarcoma in zebrafish was recently described in zebrafish with germline mutations in the zebrafish orthologue of the tumor suppressor gene PTEN.…”

“…Rhabdomyosarcoma and liposarcoma can be induced in transgenic zebrafish by targeting the Ras-Raf-MEK-ERK or PI(3)K-Akt pathways, respectively, whereas hemangiosarcoma in zebrafish is associated with haploinsufficiency for pten. 15,38,59,61 The recombination activating gene-2 (RAG2) shows specific expression in mammalian lymphoid cells, although promoter activity has been observed in nonlymphoid cell populations. 81,117 In zebrafish, the rag2 promoter is expressed in both lymphoid and nonlymphoid cell populations, including mesenchymal progenitor cells.…”

“…38 Liposarcomas were composed of well-differentiated lipocytes intermixed with atypical stromal cells and lipoblasts with pleomorphic nuclei and were locally invasive. 38 Although rhabdomyosarcoma and liposarcoma models were developed by transgenic approaches, a model for hemangiosarcoma in zebrafish was recently described in zebrafish with germline mutations in the zebrafish orthologue of the tumor suppressor gene PTEN. 15 Zebrafish have 2 copies of pten (ptena and ptenb), and 2 mutant lines carrying nonsense mutations in each gene were identified from an ENU-mutagenized library.…”

“…47,59,60 Ectopic expression of the zebrafish rag2 promoter in these mesenchymal progenitor cell compartments led to development of models for rhabdomyosarcoma and liposarcoma. 38,59 Embryonal rhabdomyosarcoma developed in zebrafish that were injected at the 1-cell stage with a human KRASG12D transgene driven by the rag2 promoter (Fig. 2).…”