Riluzole-induced apoptosis in osteosarcoma is mediated through Yes-associated protein upon phosphorylation by c-Abl Kinase

Marian Raghubir,Iffat Tariq,Lucas Cecilio,Chowdhury N Rahman,Raquel Zhagui,Salina Yan,Rifat Hasnat,Linda Wong,Sonam Lhamo,Shraddha Chandthakuri,Shahana S Mahajan,Yesmin Chowdhury,Yu Qi Huang,Angelina Blyufer,Cassey Tam,Syeda M Azeem

doi:10.1038/s41598-021-00439-8

Abstract

Our lab has previously demonstrated Riluzole to be an effective drug in inhibiting proliferation and inducing apoptosis in both human and mouse osteosarcoma. Yes-associated protein is a transcription co-activator, known to be involved in cell proliferation or apoptosis depending on its protein partner. In the present study we investigated the role of YAP in apoptosis in osteosarcoma, we hypothesized that YAP may be activated by Riluzole to induce apoptosis in osteosarcoma. By knocking down the expression of YAP, we have demonstrated that Riluzole failed to induce apoptosis in YAP deficient osteosarcoma cells. Riluzole caused translocation of YAP from the cytoplasm to the nucleus, indicating YAP’s role in apoptosis. Both Riluzole-induced phosphorylation of YAP at tyrosine 357 and Riluzole-induced apoptosis were blocked by inhibitors of c-Abl kinase. In addition, knockdown of c-Abl kinase prevented Riluzole-induced apoptosis in LM7 cells. We further demonstrated that Riluzole promoted interaction between YAP and p73, while c-Abl kinase inhibitors abolished the interaction. Subsequently, we demonstrated that Riluzole enhanced activity of the Bax promoter in a luciferase reporter assay and enhanced YAP/p73 binding on endogenous Bax promoter in a ChIP assay. Our data supports a novel mechanism in which Riluzole activates c-Abl kinase to regulate pro-apoptotic activity of YAP in osteosarcoma.

Highlights

Osteosarcoma is a primary malignant bone tumor[1], derived from mesenchymal cells that fail to differentiate into osteoblasts[2]
We have demonstrated that mGluR5, a G-protein coupled receptors (GPCRs), activates glutamate dependent growth signaling in osteosarcoma cells[8]
To determine if YAP is a key protein involved in the execution of apoptosis by Riluzole, we have knocked down the expression of YAP using lentivirus expressing shRNA against YAP

Summary

Introduction

Osteosarcoma is a primary malignant bone tumor[1], derived from mesenchymal cells that fail to differentiate into osteoblasts[2]. In stable cell lines LM7shYAP-1 lines and LM7shYAP-2 with YAP knockdown, Riluzole failed to induce apoptosis as seen by the number of DAPI positive cells, which are not significantly different from the DMSO control sample (Fig. 1A). The results, presented as percent of control sample, show that PPY A or GNF-5 did not significantly affect the viability of the cells on their own but prevented Riluzole-induced loss of viability suggesting that c-Abl kinase may be involved in Riluzole-induced apoptosis (Fig. 3A).

Results

Conclusion