Abstract A64: A role for injury in sarcomagenesis

We developed a primary mouse model of embryonal rhabdomyosarcoma and discovered that tissue injury dramatically accelerates sarcoma formation at the site of injury. We were intrigued by this finding as many sarcoma patients anecdotally report a history of trauma prior to sarcoma development. We therefore utilized this mouse model system to explore the mechanism of injury-mediated sarcoma formation. Our preliminary findings suggest that the majority of muscle progenitor cells remain quiescent following mutation of genes with high transformation potential. However, tissue injury “flips a switch”, thereby pushing resting muscle progenitor cells into a proliferative phase. In the setting of a normal mouse, proliferation of muscle cells results in resolution of the injury in a matter of weeks. Conversely, when cells in the vicinity of injury harbor transformative gene mutations, the “switch” does not return to an off-state, and a tumor rapidly develops at the injury site. Our novel mouse model of embryonal rhabdomyosarcoma provides temporospatial control over the deletion of p53 and activation of Kras in muscle progenitor cells. Specifically, Pax7-CreERT2 (P7) mice express a tamoxifen-inducible Cre downstream of the endogenous promoter for the satellite cell transcription factor Pax7. P7 mice were crossed to genetically engineered mice containing a lox-STOP-lox cassette upstream of oncogenic K-ras G12D (K) in addition to 2 floxed p53 alleles (P) to generate P7KP mice. P7KP mice were injected with systemic, intraperitoneal (IP) tamoxifen to mediate Cre-dependent recombination in Pax7-expressing cells. When treated in this manner, sarcomas arise throughout the animal with 100% penetrance with a median onset of 45 days. In contrast, tumor onset is dramatically accelerated when P7KP mice are injured with cardiotoxin, a component of cobra venom that causes myonecrosis. P7KP mice treated with IP tamoxifen along with concurrent intramuscular (IM) cardiotoxin develop sarcomas at the site of injury with a median onset of 15 days. Cardiotoxin was next injected into the gastrocnemius muscle at time points either before, concurrent, or after IP tamoxifen to better define the effect of injury on sarcoma formation. Cardiotoxin promoted highly efficient transformation when administered up to 3 days before and up to 21 days after IP tamoxifen administration. Lineage tracing studies showed no appreciable proliferation of recombined Pax7+ cells in the absence of cardiotoxin, suggesting that sarcoma formation requires factors in addition to loss of p53 and activation of K-ras to cause sarcoma. In order to test the hypothesis that proliferating satellite cells are more prone to sarcoma formation, we treated P7KP mice in the gastrocnemius muscle with hepatocyte growth factor (HGF) along with systemic, IP tamoxifen. HGF has been shown to promote proliferation of resting satellite cells. Interestingly, 12/13 mice treated with IM HGF developed sarcomas at the injection site in a median of 37 days versus 3/12 of mice treated with vehicle control. Current experiments are aimed at dissecting the role of the immune system in the cardiotoxin response. Our experiments support the role of tissue injury acting as a classic promoter in the initiator/promoter model of tumorigenesis. Thus perturbation of the microenvironment has a dramatic effect on sarcoma formation. While the majority of cancer therapeutics are directed against mutations within the tumor, our findings reveal alternative candidates to prevent sarcoma initiation and perhaps maintenance. We anticipate that an understanding of signaling events at the earliest stages of sarcoma formation will provide new drug targets to treat existing tumors and potentially prevent the outgrowth of micrometastases. Citation Format: David Van Mater, Leonor Ano, Jordan Blum, David G. Kirsch. A role for injury in sarcomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A64.