Abstract
Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA) system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues) would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively). Disrupting the G1 checkpoint CDKN2 (p16/p19−/−) resulted in sarcoma in 30% of p16/p19−/−xN-tva mice with a median latency of 23 weeks (range 8–40 weeks). A similar incidence occurred in p16/p19−/−xBKE mice (32%), however, a shorter median latency (10.4 weeks) was observed. p16/p19−/−xAKE mice also developed sarcomas (24% incidence; median 9 weeks) yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.
Highlights
Soft tissue sarcomas (STS) represent a heterogeneous group of mesenchymal tumors whose molecular genetics can be broadly divided into two categories [1]
We studied transgenic mice that express tva under the control of the promoters for nestin, aactin and b-actin to determine if certain cell types/developmental stages would be more amenable to tumorigenesis [11,12,13]
Since the CDKN2 locus, which encodes p16 (INK4a) and p19 (ARF) genes, has been implicated in human and murine sarcoma pathogenesis, we sought to determine if dysregulation of this tumor suppressor would play a cooperative role in sarcomagenesis in the RCAS-TVA system [14,22]
Summary
Soft tissue sarcomas (STS) represent a heterogeneous group of mesenchymal tumors whose molecular genetics can be broadly divided into two categories [1]. The first group is characterized by simple karyotypic defects that include recurrent chromosomal translocations and oncogenic mutations. These findings can be exploited for diagnostic confirmation and are often central to tumorigenesis. The second group is genomically unstable, resulting in complex cytogenetic lesions that characteristically harbor multiple genetic alterations, including cell cycle checkpoint mutations such as p53, CDKN2A and RB1[1]. A 4 uL mixture of DF1 chicken fibroblast cells containing oncogenic RCAS vectors was injected into the left hindlimb of neonatal mice. Staining density was scored using proportion and intensity measures as described [19]
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