The Effect And Mechanism Of Cholesterol Metabolism Reprogramming To Escape Cell Death Induced By Glutamine Restriction In Hepatocellular Carcinoma Cells

BackgroundGlutamine restriction could inhibit tumor progress,but it did not achieve the expected benefits for tumor therapy due to the heterogeneity and flexibility of tumor metabolism.It has been confirmed that when confronted with glutamine restriction,tumor cells up-regulate glutamine synthesis,increase glutamine uptake and induce autophagy to adapt to nutrient-poor conditions of the tumor microenvironment.Recent studies indicated that it plays a key role in cell survival under the glutamine restriction conditions to decrease lipogenesis and increase fatty acid β-oxidation.The liver is a major lipid metabolic organ,and lipid metabolism reprogramming contributes to the development and progression of hepatocellular carcinoma(HCC).Cholesterol,one of the most important lipids,involves in many biological processes,such as cell signal transduction,membrane building,and energy storage.However,previous studies have rarely focused on cholesterol metabolism under nutrient stress.Especially,whether glutamine restriction affects cholesterol metabolism in HCC cells is not clear yet.PurposeTherefore,we tried to clarify the effect of glutamine restriction on cholesterol synthesis in HCC cells and the underlying mechanisms.More importantly,we aimed to explore the therapeutic potential of combined glutamine restriction and cholesterol metabolism disruption in HCC.Methods and resultsWe established glutamine restricted HCC cellular and mouse models for subsequent research,including cell counting assays,RT-qPCR,Western bolt,tumor graft in nude mice,flow cytometry assays and immunofluorescence microscopy.The data presented from the HCC cellular and mouse model shows,glutamine restriction decreased the expression of HMGCR(3-hydroxy-3-methylglutaryl-CoA reductase)and SQLE(squalene epoxidase),the key enzymes of cholesterol de novo synthesis.Mechanically,glutamine restriction promoted HCC cellular lipophagy,followed by releasing cholesterol from lipid droplets that trafficked to the endoplasmic reticulum(ER)mediated by lysosomal NPC1,ultimately leading to increase ER cholesterol levels.Then the activation of SREBP2(sterol regulatory element binding transcription factor 2),the major transcriptional regulator of cholesterol de novo synthesis,was blocked by high levels of ER cholesterol.Furthermore,cellular functional assays indicated that inhibition of cholesterol synthesis in HCC cells is beneficial for cell survival under glutamine restriction.On the contrary,activation of cholesterol synthesis in HCC cells exacerbated glutamine restriction-induced cell death.We ulteriorly revealed that blocking of cholesterol transport or inhibiting autophagy enhanced the effect of glutamine restriction on anti-HCC in vitro and in vivo.Conclusion and deficiency:1.Experimental work primarily suggested that glutamine restriction induced lipid droplet autophagy,which needs more supplemental research to be confirmed;2.The underlying mechanism remains unclear in how cholesterol affects cell death induced by glutamine restriction in HCC cells.Innovation and significance:1.In this study,for the first time,we confirmed that glutamine restriction inhibits cholesterol de novo synthesis,and elucidated the underlying mechanisms in HCC.2.Inhabitation of cholesterol de novo synthesis is an adaptive strategy for HCC cells to survive under the microenvironment of glutamine restriction;3.Both blocking cholesterol transport and inhibiting autophagy could enhance the antiHCC effect of glutamine restriction.In sum,this study,as a theoretical basis,may significantly contribute to providing new strategies for the treatment of HCC under glutamine restriction microenvironment.