Targeting PKA-CREB Pathway Enhances The Inhibitory Effect Of Aspirin On Liver Cancer

Background and aims:Hepatocellular carcinoma(HCC)is a malignant tumor with high morbidity and mortality.The treatment of advanced HCC mainly relies on molecular targeted agents.Currently,sorafenib and lenfatinib are the only first-line targeted drugs available for treating HCC,but with poor long-term efficacy and frequent drug resistance.Therefore,it is necessary to explore more drugs and new strategies for treating HCC,such as repurposing of the old drugs and combining different drugs.Aspirin is the most commonly used nonsteroidal anti-inflammatory drug.Epidemiological studies have demonstrated that aspirin can reduce the risk for HCC in hepatitis B virus/hepatitis C virus carriers.Preclinical studies have shown that aspirin can inhibit the growth of HCC cells and pulmonary metastasis.We and other groups have reported that combination of aspirin and sorafenib synergistically inhibits the growth and metastasis of HCC.Given the prospect of aspirin in cancer prevention and treatment,further study of its effects on tumor cell signal transduction pathways is particularly important.Since drug resistance is a very popular problem in cancer therapy,elucidation of chemoresistance mechanisms of aspirin is crucial to developing novel therapeutic strategies to maximize the anticancer potential of aspirin.So far,little is known about the mechanisms underlying aspirin resistance.Our current study aims to investigate this critical issue.Aspirin plays an anticancer role by inhibiting COX/PGE2,Wnt/β-catenin and NF-?B,as well as activating AMPK.AMPK is an important "energy sensor" in eukaryotic cells.Previous studies have found that aspirin can regulate the p53-p21 pathway and the TSC2/raptor-m TORC1 pathway by activating AMPK.But in recent years,many studies have also demonstrated that AMPK can promote tumor progression in tumor types-or context-dependent manner,suggesting that the activation of AMPK by aspirin may have opposing effects.Blockade of the tumor-promoting effects of AMPK might enhance the anti-cancer effects of aspirin.Therefore,we need to study the mechanisms underpinning the tumor-promoting effects of AMPK and explore strategies to enhance aspirin sensitivity.The PKA-CREB pathway is abnormally activated and plays oncogenic roles in a variety of tumors,including liver cancer.AMPK is known to activate PKA,but the specific mechanisms are still unclear,while COX can activate PKA through the PGE2-EP2-AC-c AMP pathway.Given that aspirin simultaneously inhibits COX/PGE2 and activates AMPK,the effect of aspirin on the PKA-CREB pathway in HCC and its specific mechanism remain to be investigated.This study was therefore aimed to determine how aspirin affects the PKA-CREB/ATF1 axis and analyze the effect of aspirin and AMPK on s AC expression,c AMP synthesis,PKA activation and CREB/ATF1 phosphorylation,which lays the foundation for targeting the PKA-CREB/ATF1 pathway to enhance the sensitivity of HCC to aspirin.Finally,we will screen out CREB/ATF1-targeted agent and explore its combination with aspirin in treating HCC.Methods and Results:In HCC cell lines Hep G2 and Hep3 B,we use a variety of molecular biology methods,such as western blot,real-time quantitative RT-PCR(q RT-PCR),RNA interference,PKA kinase assay,detection of intracellular c AMP and so on,to study the mechanisms underlying the effects of aspirin and small molecule drug berbamine on multiple signal transduction molecules.We also studied the effects of small molecular drugs such as aspirin and berbamine or CREB/ATF1 small interfering RNA on the growth and apoptosis of HCC cells by using CCK-8 assay,Ed U labeling,colony formation experiment,cell apoptosis assay and other cell biological methods.In addition to in vitro studies,the effects of aspirin-berbamine combination on tumor growth was evaluated in HCC xenografts mouse model,and the effects of these drugs on the proliferation and expression of related proteins were detected by immunohistochemical(IHC)and western blot methods.The main results of this study are as follows:(1)Phosphorylation of AMPK and CREB/ATF1 is induced by aspirin in HCC cells Hep G2 and Hep3 B,and q RT-PCR results show that aspirin significantly increases the transcription levels of CREB and ATF1 target gene c-Jun.After inhibiting CREB/ATF1 expression with si RNA,aspirin does not up-regulate c-Jun transcription.AMPK agonist AICAR also induces CREB/ATF1 phosphorylation,and knockdown of AMPKα1 by si RNA inhibits aspirin-induced CREB/ATF1 phosphorylation.These results suggest that aspirin can promote phosphorylation and activation of CREB/ATF1 by activating AMPK.(2)By detecting the effects of aspirin in combination with MEK inhibitor U0126,AKT inhibitor MK2206,p38 MAPK inhibitor SB203580,PKA inhibitor H-89 and CAMKⅡ/Ⅳ inhibitor KN93 on CREB/ATF1 phosphorylation in Hep G2,we found that the induction of CREB/ATF1 phosphorylation by aspirin can be completely inhibited by PKA inhibitor H-89 and partly inhibited by SB203580.H-89 also completely inhibits the induction of CREB/ATF1 phosphorylation by aspirin in Hep3 B cellls.Similarly,H-89 can completely inhibit AICAR-induced CREB/ATF1 phosphorylation.(3)PKA kinase activity assay results show that aspirin increases PKA kinase activity in Hep G2 cells.ELISA results show elevated c AMP levels in aspirin-treated Hep G2 cells.Aspirin no longer induces an increase in c AMP levels after inhibiting AMPKα1 expression by si RNA.q RT-PCR and western blot results show that aspirin could induce the expression of c AMP synthase s AC in Hep G2 and Hep3 B cells.Inhibition of s AC expression with si RNA antagonizes the induction of CREB/ATF1 phosphorylation by aspirin,suggesting that aspirin activates the PKA-CREB/ATF1 pathway by inducing s AC expression and promoting c AMP synthesis.(4)s AC expression and activation can be promoted by bicarbonate.Further studies show that the bicarbonate neutralizer lactic acid could antagonize the aspirin-induced CREB/ATF1 phosphorylation in Hep G2 and Hep3 B cells.q RT-PCR assays show that aspirin has no effect on the transcriptional levels of carbonic anhydrase CA2,CA9 and CA12,which mediate the generation of bicarbonate.Therefore,we further studied the effect of aspirin on the urea cycle enzyme carbamoyl phosphate synthetase1(CPS1)that is involved in the consumption of bicarbonate and ammonia to generate carbamoyl phosphate.(5)q RT-PCR results show that aspirin could down-regulate CPS1 transcription in Hep G2 and Hep3 B cells.Western blot results show that aspirin inhibit the expression of CPS1,and AICAR also reduce the expression of CPS1.Knockdown of AMPKα1 by si RNA antagonizes the down-regulation of CPS1 and up-regulation of s AC by aspirin.(6)After inhibiting CPS1 expression with si RNA in Hep G2 and Hep3 B cells,s AC expression and CREB/ATF1 phosphorylation levels are increased.Inhibition of CPS1 expression also results in increased intracellular c AMP levels and enhanced PKA kinase activity.(7)Aspirin and PKA inhibitor H-89 synergistically inhibit Hep G2 and Hep3 B cell growth.Similarly,CREB/ATF1 knockdown and aspirin also synergistically inhibit the growth of Hep G2 and Hep3 B cells,suggesting that both PKA and CREB/ATF1 antagonize the inhibitory effect of aspirin on HCC cells.(8)Drug screening demonstrates that the natural product berbamine and aspirin could synergistically inhibit the growth of HCC cells.Colony formation experiments show that the combined treatment of aspirin and berbamine significantly inhibit the colony formation ability of Hep G2 cells.Ed U assay show that aspirin-berbamine combination significantly inhibits Hep G2 and Hep3 B cell proliferation.Hoechst33342 staining shows that the combination of aspirin and berbamine does not affect cell apoptosis,and neither the caspase inhibitor Z-VAD-FMK nor the necrosis inhibitor necrostatin-1 affects the inhibition of HCC cells growth by aspirin-berbamine combination.(9)Western blot and PKA kinase activity assay show that berbamine could antagonize the induction of CREB/ATF1 phosphorylation by aspirin,but does not affect PKA kinase activity.In addition,inhibition of CAMK2 G,a known target of berbamine,does not affect the induction of CREB/ATF1 phosphorylation by aspirin and the effect of aspirin on Hep G2 cell growth.Western blot analysis shows that the PP2 A inhibitor Okadaic acid could antagonize the inhibition of aspirin-induced CREB/ATF1 phosphorylation by berbamine,suggesting that berbamine inhibits the phosphorylation of CREB/ATF1 through CAMK2G-independent and PP2A-dependent pathways.(10)Aspirin and berbamine combination can significantly inhibit the expression of cyclin D1 in Hep G2 and Hep3 B cells,and CREB/ATF1 knockdown in combination with aspirin can also significantly reduce the expression of cyclin D1.Ed U staining shows that CREB/ATF1 knockdown in combination with aspirin significantly reduces the proportion of Ed U-positive cells in Hep G2 and Hep3 B cells.(11)Tumor xenografts experiments in nude mice show that the combination of aspirin and berbamine could significantly inhibit the growth of Hep G2 xenografts compared with either agent alone.IHC analysis of PCNA shows that the combination of aspirin and berbamine significantly inhibits the proliferation of HCC cells.IHC analysis also shows that aspirin could significantly reduce the expression of CPS1 in tumor tissues.Western blot analysis shows that aspirin does not affect CPS1 expression or CREB/ATF1 phosphorylation in normal mouse liver tissues.Conclusion:The results of this study suggest that aspirin induces the phosphorylation and activation of CREB/ATF1 in HCC cells by activating AMPK.Aspirin down-regulates the expression of CPS1 through AMPK and reduces the consumption of bicarbonate,thereby activating the s AC-c AMP-PKA-CREB /ATF1 pathway.Therefore,in addition to participating in cell metabolism,CPS1 also regulates the c AMP-PKA-CREB /ATF1 signaling pathway.Targeted inhibition of the PKA-CREB/ATF1 pathway can enhance the sensitivity of HCC cells to aspirin.The natural product berbamine can inhibit the induction of CREB/ATF1 phosphorylation by aspirin in PP2A-dependent manner,and enhance the sensitivity of HCC cells to aspirin.The combination of aspirin and berbamine synergistically inhibit the growth of HCC cells and tumors,and this strategy holds promise in treating HCC.