Mechanistic Study Of AMPK In Maintaining Tumor Cell Survival

Tumor is a genetic disease with a complex and highly heterogeneous etiology,but has some common characteristics,such as the Warburg effect and addiction to certain genes(gene addiction).Gene addiction,including oncogene addiction and non-oncogene addiction,is a phenomonen that tumor cells increase dependences on the function of certain genes.These dependences play key roles on cell growth and survival and are difficult to overcome in the short term.AMP-activated protein kinase(AMPK)is an energy and nutrient sensor that regulates cellular energy homeostasis and metabolic balance.The traditional view of AMPK is a tumor suppressor that can inhibit tumor progression by metabolical regulation or stabilizing other tumor suppressors,such as p53,TET2,and TSC2,in early stage of tumorigenesis.However,there is increasing evidences that AMPK is overexpressed or overactivated in a variety of tumor cells.Blocking AMPK signaling in such types of tumor cells can significantly inhibit cell growth or even induce cell death,suggesting AMPK kinase addiction(non-oncogene addiction)developed.For such AMPK-addicted tumors,AMPK may serve as a potential therapeutic target.However,this view still lacks in-depth research and conclusive experimental evidence so far.Herein,we systematically study the role and molecular mechanism of AMPK in maintaining the survival of tumor cells,including:(1)Effects of AMPK on the viability and survival of tumor cells.Firstly,we analyzed the expression and activation of AMPK in some tumor samples and cell lines by bioinformatics,and found AMPK was significantly upregulated in a variety of hematological cancers;Secondly,we analyzed the relationship between AMPK expression and prognosis of cervical cancer patients,found patients with high AMPK expression had a lower 5-year survival rate;Thirdly,we treated Jurkat,K562,Hela and primary cancerous B cells with Dorsomorphin and found AMPK blockade by Dorsomorphin treatment markedly impaired cell viability,and promoted cell death throuth AMPK-dependent manner.Taken together,AMPK was upregulated and required for tumor cell survival.(2)Effects of AMPK on the growth of Hela xenografts in nude mice.To study the roles of AMPK in the survival of tumor cells in vivo,we created the Hela xenograft mouse model and treated them with Dorsomorphin through intraperitoneal injection.We found that AMPK blockade by Dorsomorphin can significantly suppress the xenografts growth in vivo(3)Study on the molecular mechanisms of AMPK inhibition-mediated cell death To explore the apoptotic networks activated upon AMPK blockade,we performed an RNA-seq analysis to compare the transcriptome of Dorsomorphin-treated K562 cells with control cells and found BAD up-regulation and proteostasis collapse may be the main mechanisms of AMPK blockade-induced tumor cell death.Supportive of the transcriptomic data,realtime PCR and Western blot analysis of Dorsomorphin-treated cancer cells confirmed markedly upregulation of BAD mRNA and protein,misfolded protein accumulation,along with activation of Caspase 3,an important effector caspase that executes apoptosis.Moreover,BAD knockdown partially relieves AMPK inhibitor-induced tumor cell death.These results suggest that AMPK maintains tumor cell survival mainly through the regulation of BAD expression and protein homeostasis.(4)Blockade of AMPK activity enhaces the anti-tumor activity of proteasome inhibitor PS-341.Previous in vitro study revealed that Dorsomorphin significantly promotes misfolded protein accumulation in tumor cells,leading us to speculate that AMPK blockade may promote proteasome inhibitor-induced cell death.Thus,we treated tumor cells and Hela xenografts with Dorsomorphin,PS-341(Protein inhibitor),or in combination and found Dorsomorphin-mediated AMPK blockade markedly promotes PS-341-mediated cell death in vitro and enhaces the anti-tumor effects of PS-341 in vivoIn summary,this study confirms that AMPK plays important roles in maintaining tumor cell survival through two ways as below:first of all,AMPK can promote tumor cell survival by regulating the expression of BAD,a well-known pro-apoptotic protein secondly,AMPK also helps tumor cells to maintain protein homeostasis,a process crucial for cell survival.Therefore,Blockade of AMPK activity significantly promotes BAD expression,proteostasis collapse in tumor cells,and promoted proteasome inhibitor PS-341-induced tumor cell death in vitro and enhances its anti-tumor effects in vivo.These findings not only broaden our understanding of AMPK pathological functions,but also provide new targets,ideas and theoretical guidance for clinical tumor therapy.