ANGPTL4 Negatively Regulate the Progression of Osteosarcoma by Remodeling Branched-Chain Amino Acid Metabolism

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Background: : Angiopoietin-like-4 (ANGPTL4), a secreted glycoprotein that is mainly recognized as a regulator in lipid metabolism, now, is implied in the regulation of the growth and metastasis of various carcinomas. However, less is known about its functions in the progression of sarcomas, let alone osteosarcoma (OS), which is the most common malignant diagnosed in musculoskeletal system. Methods: : The expression of ANGPTL4 in clinical OS samples and cell lines paired with their controls were analyzed in both mRNA and protein levels. Cell functional analysis including proliferation and colony formation were carried out to detect the roles ANGPTL4 takes in the progress of OS using stable ANGPTL4 overexpression and knockdown HOS/MNNG cell lines. The RNA-Seq and bioinformatics analysis were then employed to discover the BCAA metabolism related signaling which is involved in ANGPTL4 functioning on HOS/MNNG cell growth. Furthermore, BCAAs content measurement, and BCATs rescue experiments were performed to confirm the BCAA/mTOR signaling axis that ANGPTL4 triggered in HOS/MNNG cells. Finally, a xenograft mouse model was carried out to further verify the ANGPTL4 /BCAA/mTOR signaling axis discovered. Results: : We found that the expression of ANGPTL4 is reduced in clinical OS tissues and cell lines compared to cancellous bone tissues and BMSCs, respectively. The knockdown of ANGPTL4 in HOS/MNNG cells results in enhanced cell growth and clone formation. Moreover, BCAA/mTOR signaling axis were discovered to be triggered by ANGPTL4 down regulation in HOS/MNNG cell using RNA-seq. It was also verified that the accumulation of BCAAs activates the mTOR signaling pathway, and in turn promotes HOS/MNNG cell growth using BCAAs content measurement, and BCAT inhibition. Finally, the IHC results of xenograft mouse model also confirmed this ANGPTL4/BCAA/mTOR signaling axis in vivo . Conclusions: : Taken together, our results demonstrate that the expression of ANGPTL4 were negatively related to OS progress. Moreover, it was found the down-regulation of ANGPTL4 promoted OS cell growth via BCAAs/mTOR axis.