siRNA Knockdown of RPTOR Reduces RPTOR Expression, Increases BRD4 Expression, and Alters Proliferation and Apoptosis in Ewing's Sarcoma Cells

Authors

• Sergio Cipriano, St Marys UniversityFollow
• David Leavitt, St Marys UniversityFollow
• Michael Olivia, St Marys UniversityFollow
• Liam Valdez, St Marys UniversityFollow
• Terry Jo Shackleford, St Marys UniversityFollow

Files

Publication Date

Spring 5-10-2026

Keywords

Ewing Sarcoma, Pediatric Cancer, RPTOR, mTOR, FGFR1, BRD4, LY2874455, Apoptosis, Proliferation, qRTPCR, Incucyte, Tissue Culture

Description

Ewing sarcoma is a highly aggressive cancer that primarily affects children and young adults. Although treatment options exist, many patients do not respond effectively, showing the need for improved targeted therapies. RPTOR is a key in mTORC1 complex which regulates cell growth, proliferation, and survival. LY2874455 is a selective pan-FGFR inhibitor that targets growth factor signaling pathways involved in tumor progression, and FGFR signaling interacts with pathways such as mTOR, making it a potential target for combination therapies. We hypothesized that silencing RPTOR in Ewing sarcoma cells would disrupt mTOR signaling and alter expression of genes linked to cell survival and signaling pathways. To test this, cells were transfected with siRPTOR or siControl, followed by RNA purification, cDNA synthesis, and qRT-PCR analysis of RPTOR, FGFR1, and BRD4 expression normalized to HPRT1. RPTOR expression was significantly reduced confirming effective gene silencing. FGFR1 expression showed minimal change (1.10-fold), while BRD4 expression increased (1.9-fold), showing activation of compensatory signaling pathways. Cell proliferation and apoptosis were further assessed using Incucyte live-cell imaging. LY2874455 treatment reduced proliferation and increased caspase 3/7 activity in control cells, while siRPTOR-treated cells showed reduced apoptotic response. Overall, these findings demonstrate successful RPTOR knockdown and suggest that disruption of mTOR signaling may activate alternative pathways that influence cell survival, highlighting the complexity of targeting this pathway in Ewing sarcoma.

Collection

Cell and Molecular Methods

Format

pdf

Size or Duration

13 Pages

City

San Antonio

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

Recommended Citation

Cipriano, Sergio; Leavitt, David; Olivia, Michael; Valdez, Liam; and Shackleford, Terry Jo, "siRNA Knockdown of RPTOR Reduces RPTOR Expression, Increases BRD4 Expression, and Alters Proliferation and Apoptosis in Ewing's Sarcoma Cells" (2026). Cell and Molecular Methods. 9.
https://commons.stmarytx.edu/biostulab25/9