The Mechanisms Of CIP2A Alone And In Combination With PTEN Deficiency In Lung Tumorigenesis/Screening For And Mechanisms Of ACE2 Small Molecule Inhibitors In Lung Epithelial Cells

There are 2.2 million new lung cancer cases and 1.8 million lung cancer deaths worldwide each year.Lung cancer ranks No.1 in the world and in China for cancer deaths.In the past two decades,lung cancer research has continued to develop.The use of small molecule tyrosine kinase inhibitors and immunotherapy has significantly prolonged the patient’s survival period and improved the patient’s quality of life.However,these therapies are only effective for a small number of patients,and resistance is inevitable.The overall cure and survival rates for lung cancer remain low,and the 5year survival rate is only 21%.Therefore,it is still an urgent requirement to use a suitable lung cancer model to analyze the mechanism of lung tumorigenesis,and to find new key carcinogenic factors to develop targeted therapeutic drugs.CIP2A(cancerous inhibitor of protein phosphatase 2A)has been regarded as a very important oncoprotein in recent years.Previous studies have shown that CIP2A is highly expressed in a variety of malignant tumors and is closely related to the malignant transformation of cells,but its role and mechanism in the occurrence of lung cancer have not been elucidated.In this study,we found that CIP2A was highly expressed in nonsmall cell lung cancer(NSCLC)and correlated with patient prognosis.CIP2A promotes tumor cell proliferation in cell lines.Previously our team used the pulmonary surfactant protein C(SPC)gene promoter to establish a transgenic C57BL/6 mouse with a specific overexpression of CIP2A in type Ⅱ alveolar epithelial cells.In this study,SPC-CIP2A mice were bred and monitored with Micro-CT.We found that SPC-CIP2A transgenic mice developed lung cancer at 12-15 months with an incidence of 20.2%.By multiomics analysis and Seahorse metabolic assay,we found that oxidative phosphorylation in lung tumor tissues of mice with high CIP2A expression was significantly enhanced.The phosphorylation microarray results suggested that CIP2A significantly enhanced the phosphorylation of FOXO3A.Co-immunoprecipitation and immunofluorescence experiments demonstrated that FOXO3A interacts with PP2A subunit B56α.By isolating mitochondria,nucleus and cytoplasm,we found that inhibition of B56α by CIP2A enhanced FOXO3 A phosphorylation,which in turn altered the subcellular localization of FOXO3A.We next stably knocked out the FOXO3A gene in cell lines by the CRISPRCas9 system,exogenously expressing FOXO3A mutants with nuclear localization sequence deletion(ΔNLS)and impaired mitochondrial localization(Δ1-153aa).We found that altered subcellular localization of FOXO3A promotes non-small cell lung cancer(NSCLC)cell proliferation.In order to increase the lung cancer incidence and accelerate the lung cancer onset process,we selected the gene knockout mouse models for tumor suppressor genes TP53,RB1,STK11,and PTEN,which have a high mutation rate in NSCLC,to test the combined effects of CIP2A overexpression and deficiency in the above genes in initiating lung cancer.We found that CIP2A overexpression and PTEN deficiency exerted synergistic carcinogenic effect,in that lung cancer incidence in Pten-/-mice was 7.7%,but the lung cancer incidence in CIP2A/Pten-/-mice reached 53.8%.We found that CIP2A/Pten-/-led to enhanced FOXO3 A mitochondria localization compared with CIP2A overexpression and PTEN-/-groups.In TCGA database,lung adenocarcinoma(LUAD)patients with high expression of CIP2A and low expression of PTEN had poor prognosis.The CIP2A/PTEN score can be used as independent prognostic factors for patients’ overall survival and progression-free survival interval.Taken together,this study revealed that CIP2A enhances oxidative phosphorylation by regulating the subcellular localization of FOXO3A to induce lung cancer.CIP2A overexpression and PTEN deficiency induced potentiated lung carcinogenic effects,providing a new idea for the treatment of lung cancer.Angiotensin-converting enzyme 2(ACE2)is the receptor of the severe acute respiratory syndrome coronavirus 2(SARS CoV-2),the pathogen of COVID-19.SARSCoV-2 infects host cells through the Spike(S)glycoprotein acting on the ACE2 receptor.The S protein,especially its receptor binding domain(RBD),is often mutated,causing antibodies to fail to recognize it,thereby evading immune surveillance.However,the ACE2 sequence is rarely mutated,and inhibiting the ACE2 receptor can effectively reduce the infection of cells by SARS CoV-2.Therefore,targeting ACE2 is a promising target for the development of therapeutic drugs.In this study,in order to find inhibitors that can modulate ACE2 expression to prevent SARS-CoV-2 from entering lung epithelial cells,we used small molecule compound libraries for screening.First,ACE2 promoter-luciferase constructs were transiently transfected into HLF cells and luciferase assays were performed.The results showed that among the 64 natural compounds tested,6-O-angeloylplenolin(6-OAP)exhibited the most potent inhibitory activity on ACE2 expression.6-OAP was identified as one of the most abundant sesquiterpene lactones extracted from medicinal herb Centipeda minima(CM).Therefore,we used qPCR to verify that 6-OAP and CM can inhibit ACE2 transcription.To further explore the specific mechanism of 6-OAP regulating ACE2 transcription,chromatin immunoprecipitation assay and electrophoretic mobility shift assay found that 6-OAP(2.5μM)significantly inhibited the interaction between Stat3 protein and ACE2 promoter.In vitro,western blot experiments confirmed that 6-OAP(1.25-5μM)and its parent medicinal material CM(0.125%-0.5%)can dose-and timedependently down-regulate ACE2 in normal lung epithelial cells 16HBE and Beas-2B.Meanwhile,consistent results were observed in the lung tissue of C57BL/6 mice after administration of 6-OAP(50mg/kg/d)or CM(0.5%)for one month.Furthermore,we found that 6-OAP/CM dose-dependently reduced IL-6 production in 16HBE cells,and down-regulated chemokines such as CXCL13,CX3CL1,and CXCL11.Meanwhile,we found that 6-OAP/CM can effectively inhibit SARS-CoV-2 S protein pseudovirus entry into target cells,suggesting that 6-OAP/CM may potentially protect lung epithelial cells from SARS-CoV-2 infection.Taken together,the study revealed that 6-OAP and its parent herb CM reduced ACE2 expression and blocked SARS-CoV-2 S protein pseuduovirions infection through suppression of TF STAT3.Therefore,as an inhibitor of ACE2,CM/6-OAP provides a new idea for the prevention and treatment of COVID-19.