The Role Of Asparagine Synthetase In Ovarian Cancer And Its Expressional Regulation

Ovarian cancer is gynecological malignant tumor with the highest mortality rate,which seriously threatens women's life and health.Up to now,a large number of studies have reported that asparagine synthetase(ASNS)plays a key role in the development of various types of malignant tumors,and its role in ovarian cancer has not been studied.Therefore,this paper explores the role and expressional regulation of ASNS in ovarian cancer.ASNS is ubiquitous in mammalian organs and its main biological function is the synthesis of asparagine by glutamine.Studies have shown that asparagine has an irreplaceable role in regulating cell adaptation to glutamine deprivation.Most tumor cells often have glutamine addiction,and glutamine metabolism plays a crucial role in the development of tumors.As the rate-limiting enzyme of glutamine metabolism,glutaminase(GLS)has become a target for cancer therapy.Therefore,GLS inhibitors have become a research hotspot.Our study found that GLS inhibition or knockdown can upregulate ASNS in ovarian cancer cells.So,is there a connection between GLS and ASNS?What is the expressional regulation of ASNS?In addition,our experimental results have shown that ASNS has a tumor-promoting effect in ovarian cancer.Does the dual blockade of asparagine synthesis and glutamine metabolism have a better therapeutic effect on ovarian cancer?Therefore,we explored the above questions,carried out the following work,and obtained a series of results:1.The different expression of ASNS in ovarian cancer tissues and cell linesThe expression levels of ASNS in ovarian cancer were analyzed by real-time quantitative PCR and Western blotting.The results showed that the expression of ASNS was different in different ovarian cancer patients and different types of ovarian cancer cell lines.TCGA database analysis showed that the expression of ASNS was correlated with the prognosis and that the disease-free survival of ovarian patients with low expression of ASNS was significantly prolonged.2.ASNS promotes tumor growthThe results of MTT and clony formation showed that knockdown of ASNS significantly inhibited the proliferation of ovarian cancer cells,indicating that ASNS played a pro-tumor role in ovarian cancer.Exogenous supplementation of asparagine could rescue the proliferation inhibition caused by ASNS knockdown,but not glutamate,suggesting that the proliferation inhibition caused by ASNS knockdown was due to the lack of asparagine and asparagine plays a critical role in the proliferation of ovarian cancer.Flow cytometry analysis of the cell cycle distribution showed a G1 phase arrest of A2780 cells with ASNS knockdown,and the exogenous addition of asparagine could reverse this phenomenon,while ASNS knockdown had no effect on cell cycle distribution in HO8910 cells.Therefore,ASNS knockdown inhibited cell proliferation in different ways in different cell lines.ASNS knockdown inhibited cell proliferation by inducing cell cycle arrest in A2780 cells,while the mechanism of proliferation inhibition in HO8910 cells with ASNS knockdown has remains to be clarified.3.Glutamine,the substrate of ASNS,upregulates ASNSThe expression of ASNS was measured by real-time quantitative PCR and Western blotting in ovarian cancer cells treated with GLS inhibitor CB-839.The results showed that the expression of ASNS at mRNA and protein levels was significantly increased after the inhibition of glutamine metabolism.At the same time,GLS knockdown with small interfering RNA also showed an increase in expression of ASNS.In addition,treatment of ovarian cancer cells with glutamine-free medium resulted in a rise at ASNS mRNA levels,while protein levels were decreased,showing an opposite trend.The analysis of the possible reasons was that ATF4 was upregulated via amino acid reaction after glutamine deprivation,which raised the ASNS mRNA levels.The protein levels of ASNS is decreased due to the reduced substrate or inhibition of protein synthesis.The above results showed that as a key substrate of ASNS,glutamine levels affected the expression of ASNS.4.Asparagine,the product of ASNS,downregulates ASNSThe expression of ASNS was measured by real-time quantitative PCR and Western blotting after exogenous asparagine supplementation.The results showed that the increased asparagine levels inhibited the expression of ASNS.Exogenous supplementation of asparagine could partially reverse upregulation of ASNS caused by GLS inhibition.As another product of the ASNS catalytic reaction,glutamate does not affect the expression of ASNS and does not reverse the upregulation of ASNS caused by GLS inhibition.When GLS activity is inhibited,tumor cells may synthesize a large amount of asparagine by glutamine by upregulating ASNS.5.The double blockade of asparagine synthesis and glutamine metabolism inhibits tumor growthThe results of MTT and clony formation showed that the combination of ASNS knockdown and GLS inhibitor can enhance the proliferation inhibition effect on ovarnan cancer cells.Flow cytometry analysis of the cell cycle distribution found that ASNS knockdown combined with GLS inhibitor further induced a G1 phase arrest of A2780 cells,but still had not affect on cell cycle distribution in HO8910 cells.At the same time,ROS levels were evaluated to be elevated in ovarian cancer cells induced by the combination of ASNS knockdown and GLS inhibitor.Due to the heterogeneity of ovarian cancer cells,the pathways of cell proliferation inhibition is different.The inhibition of proliferation in A2780 cells is mainly through the induction of cell cycle arrest,possibly by inducing ROS elevation,while the inhibition of proliferation in HO8910 cells may be through inducing ROS elevation.In conclusion,this paper demonstrated a role of ASNS in ovarian cancer,explored the effects of glutamine and asparagine on the expression of ASNS.We proposed the possibility of treatment of ovarian cancer with the combination of ASNS inhibition and GLS inhibition,which provides new directions and strategies for the treatment of ovarian cancer,and contributes to the discovery and application of new biomarkers and therapeutic targets.