Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence

Jenny J Li,Candy Chen,Kristianne M Oristian,Alexander R Kovach,Margaret Demonia,Corinne M Linardic,Katherine K Slemmons

doi:10.1038/s41598-021-95355-2

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level—eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a “quiet” genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.

Highlights

Rhabdomyosarcoma is a malignant tumor of mesenchymal origin characterized by skeletal muscle histogenesis
We investigated the role of RAS in Rh28 aRMS cells, since RAS mutations are common in eRMS, they are rare in aRMS cells expressing PAX3-FOXO1
We reasoned that either oncogenic RAS is not required for tumorigenesis of aRMS or is not tolerated in aRMS

Summary

Introduction

Rhabdomyosarcoma is a malignant tumor of mesenchymal origin characterized by skeletal muscle histogenesis. RAS proteins carry out these functions by acting as molecular switches, alternating between the active GTP-bound and the inactive GDP-bound states While their function is tightly regulated in normal cells, oncogenic RAS mutations yield constitutively active proteins that stimulate uncontrolled proliferation and survival in tumor cells[6]. First described in 1997, OIS occurred when oncogenic RAS was expressed in rodent or human fibroblasts, leading to permanent growth arrest indistinguishable from replicative cellular s enescence[12]. This growth arrest was associated with increases in the levels of the DNA-damage response sensor p53 and the cyclin dependent kinase inhibitors p21 and p16 and decreases in the levels of phospho-RB12. Of the three main RAS effector pathways (MAPK/ERK, PI3K/AKT, RALA/B), the MAPK/ERK and the PI3K/AKT pathways play crucial roles in O IS14,19–21

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