Abstract
Purpose :
Retinoblastoma tumours(Rb) are caused by loss of the Rb protein due to mutations. Rb tumors can flexibly use glutamate for energy and growth. BCAT1 (branched chain amino acid transferase 1) catalyzes BCAA (branched chain amino acids) into their respective branched chain keto acids towards glutamate. Autophagy dysregulation has been reported in Rb and BCAA are known to regulate mTORC1. We therefore investigate BCAT1 function in tumor growth in the context of regulating BCAA and autophagy in Rb.
Methods :
Levels of BCAA and glutamate were measured in vitreous humor of Rb containing eyes collected at enucleation. Expression of BCAT1, E2F2, etc were evaluated by immunohistochemistry in tumor tissues. The Rb-/- WERI-RB1 cells were transduced with BCAT1 overexpression or BCAT1 targeting Crispr-Cas9 constructs to establish gain or loss-of-function models of BCAT1 to evaluate proliferation and 3D tumor formation. WERI-RB-1 cells were treated with Trehalose, Chloroquine, etc., to modulate autophagy pathways. The effect of BCAT1 expression modulation and autophagy modulation in WERI-RB1 cells were analyzed by immunoblotting and gene expression analysis.
Results :
The ratio of glutamate/BCAA levels were significantly higher in vitreous humor of Rb eyes compared to healthy controls (p<0.05). IHC analysis from Rb patient tumors revealed BCAT1 and E2F2 upregulation compared with pediatric control retina. Ectopic BCAT1 expression in WERI-RB1 cells enhanced cell proliferation and 3D spheroid formation (p<0.05). BCAT1 ablation displayed reduction in proliferation (p<0.05) similar to wtRB1 over-expressing cells. BCAT1 reduction also modulated mTORC1 phosphorylation, increased LAMP1, lipidation of LC3, indicative of autophagy activation. Treatment of WERI-RB1 cells with Trehalose or Rapamycin phenocopied the effects of BCAT1 ablation with reduced proliferation and autophagy activation.
Conclusions :
Conversion of BCAA to glutamate in Rb eyes supports enhanced BCAT1 function. Lack of Rb protein shifts metabolism of OXPHOS, which is supported by glutamate availability. Thus, BCAT1 expression supports enhanced proliferation and additionally prolongs tumour sustainability by reducing autophagy which is reversed by Trehalose and Rapamycin treatment. In summary, RB tumour upregulate BCAT1 to procure alternate fuels and inhibit autophagy mediated cell death.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.