Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation.

Timothy Mckinnon,Leah Kabaroff,Marielle E Yohe,Manon Alkema,Rebecca A Gladdy,Rosemarie Venier,Brendan C Dickson,Li Chen,Jack F Shern,Javed Khan

doi:10.18632/oncotarget.3856

Abstract

While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53-/- myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53-/- myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum.

Highlights

Soft tissue sarcomas (STS) are the most common extracranial solid tumors in childhood and rhabdomyosarcoma (RMS) is the most frequent STS in this population [1]
To create mosaic sarcoma mouse models, skeletal muscle progenitors were isolated from p53-deficient mice as p53 is a known tumor suppressor in sarcoma and myoblasts are candidate cell of origin for RMS [19]
Despite recent reports in defining the genetic landscape of pediatric sarcomas, survival rates for patients in high-risk categories have not changed in the past decade as it is still unknown which molecular events are key for www.impactjournals.com/oncotarget sarcomagenesis [1]

Summary

INTRODUCTION

Soft tissue sarcomas (STS) are the most common extracranial solid tumors in childhood and rhabdomyosarcoma (RMS) is the most frequent STS in this population [1]. The genetic landscape of human RMS has identified possible oncogene or tumor suppressor gene combinations that may contribute to rhabdomyosarcoma initiation and progression [4]. Mosaic mouse models involve engraftment of host tissue with genetically manipulated progenitor cells and can be orthotopic and syngeneic. The use of tissue-specific progenitors as the cell of origin and the relative ease of genetic manipulation in donor cells allows for mosaic models to be employed in oncogenomic-based screens to functionally validate genetic data from human tumors. In a recent study investigating the origins of RMS using developmentally restricted murine models, ERMS arose from Myf6-expressing, differentiating myoblasts with inactivated tumor suppressor genes, Trp or Ptch1 [19]. To determine whether cellular transformation of myoblasts can result in RMS, we created novel mosaic mouse models, which involved orthotopic injection and engraftment of p53−/− myoblasts into syngeneic hosts. Myoblasts were selected using fluorescence activated cell sorting (FACS) and injected into syngeneic mice, resulting in rapid formation of high-grade sarcomas with myogenic differentiation

RESULTS

DISCUSSION

MATERIALS AND METHODS