The Role And Mechanism Of Iron-dependent CDK1 In Lung Carcinogenesis

Purposes: Lung cancer is the second most common cancer and the leading cause of cancer-related mortality among men and women worldwide.As we all know,Lung carcinogenesis is closely associated with smoking,occupational exposure,environmental pollution,ionizing radiation,genetics and other factors.However,the underlying mechanism of lung carcinogenesis has not yet been elucidated.More factors that might promote lung carcinogenesis and the underlying molecular mechanisms remain to be explored.A better understanding of the molecular mechanism underlying lung carcinogenesis would contribute to the development of novel strategies for its prevention and targeted therapy.Iron is an essential nutrient.Altered iron metabolism is closely associated with cancer initiation and progression.Epidemiological studies have reported that high dietary iron intake significantly elevate the risk of several malignancies,including esophageal cancer,colorectal cancer,liver cancer,and lung cancer.Amounts of experimental studies data are consistent with epidemiological data.Altogether,these studies suggested the important role of iron in lung carcinogenesis;however,the underlying molecular mechanism remains unknown.Cyclin-dependent kinases(CDKs)are critical drivers of cell cycle transition.In particular,CDK1 is a key determinant of mitotic progression,and its activity is dysregulated via indirect genetic alteration in tumorigenesis.Integration of gene expression data from TCGA(The Cancer Genome Atlas)and GEO(GENE EXPRESSION OMNIBUS)demonstrated CDK1 upregulation in lung adenocarcinoma.Furthermore,CDK1 upregulation is associated with a poor prognosis.Selective targeting of CDK1 might constitute a novel strategy for tumor treatment in certain contexts.However,the molecular mechanisms and potential applications of CDK1 in lung cancer remain undetermined.In the context of lung cancer,the study of the role and mechanism of CDK1 would help to explore the potential application methods.The inflammatory response plays a crucial role in the development and progression of tumors.L-6 and related cytokines are critical lynchpins between inflammation and cancer.Systemic and pulmonary IL-6 levels are reportedly elevated in patients with lung adenocarcinoma and these elevations are associated with poor patient survival.IL-6 promotes KRAS-driven lung carcinogenesis,and genetic ablation of either IL-6 or STAT3 suppresses the extent of lung cancer.The STAT3 signaling pathway is one of the major downstream pathways for IL-6 activation.When IL-6 binds IL-6Ra,GP130 dimerizes and activates JAK/STAT3 signaling.Lots of studies have reported that constitutive activation of STAT3 signaling is a common feature in lung cancer,and STAT3 signaling is closely associated with tumor cell proliferation,anti-apoptosis,immune evasion,and stemness.Evidences above indicates that IL-6/STAT3 signaling pathway is also closely associated with lung carcinogenesis.In summary,iron,CDK1,IL-6/STAT3 signaling pathway might play an important role in lung carcinogenesis.However,whether there is a closely correlation among iron,CDK1 and IL-6/STAT3 signaling in the process of lung carcinogenesis,has not been determined.It was recently reported that iron-dependent CDK1 promotes colorectal tumorigenesis via activating the JAK/STAT3 signaling pathway.This study aimed to investigate the role of iron/CDK1/STAT3 signaling in lung carcinogenesis.Our study is mainly divided into three parts:1.The effect of iron,CDK1,STAT3 on the colony formation and sphere formation in lung cancer cells;2.The molecular mechanism underlying on the activation of STAT3 signaling by iron-dependent CDK1;3.The role and mechanism of irondependent CDK1 in human lung tumor xenograft.Method: 1.The effect of iron,CDK1,STAT3 on the colony formation and sphere formation in lung cancer cells This part of the study mainly performed soft agarose colony formation assay and lowadherent 96-well plates sphere culture assay.The number of colony formation and the size of the sphere reflect the stemness and the ability of tumorigenesis in lung cancer cells.We respectively used lentiviral sh RNA to knock down the expression of NS(non-specific sequence),CDK1,and STAT3 in human lung adenocarcinoma cell lines A549 and 1792.FS(ferrous sulfate)was added to the soft agarose to compare the number of colony formation;FS,iron chelator DFO(Deferoxamine),CDK1 inhibitor RO3306(CDK1i)and the STAT3 inhibitor(Stattic)was added to the culture medium in the sphere culture assay.Then we observed and compared the sphere size among different groups.2.The molecular mechanism underlying on the activation of STAT3 signaling by irondependent CDK1 In this part of the study,human lung cancer cells A549 and 1792 were treated with FS,CDK1 i,JAK inhibitors Roxolitinib(JAKi),IL-6,DFO,respectively;or si RNA was used to knock down NC(Non-specific control,non-specific control),GP130,CDK1,treated with FS or not,Western blot analysis the expression of GP130,p-STAT3 and other proteins to investigate the effects of various intervention conditions;We used q PCR(Quantitative real-time polymerase Chain reaction)to detect the expression level of corresponding m RNA in cells;We used protein translation inhibitor CHX to block protein translation,treated with FS or CDK1 i to assess the degradation rate of GP130 under different conditions;we used proteasome-dependent degradation inhibitor MG132 and lysosome-dependent degradation inhibitor CHL,and analysis whether it could restore CDK1 i or si CDK1-induced decrease in GP130 expression;We treated cells with capdependent translation inhibitor 4E1 RCat,and analysis whether GP130 is a cap-dependent translational protein;Western blot analysis was used to analysis the expression of p-4EBP1(S65/T70)and other proteins,and determined whether iron-dependent CDK1 play an important role on the phosphorylation of 4E-BP1;in vitro phosphorylation assay was performed to determine whether iron has an effect on phosphorylation of 4E-BP1 by CDK1/Cyclin B1;in vitro protein translation assay was performed to detect the ability of protein translation under the different conditions.3.The role and mechanism of iron-dependent CDK1 in human lung tumor xenograft In this part of the study,lung cancer cells injected subcutaneously in nude mice.A549-NS/sh CDK1 cells were injected subcutaneously into nude mice to observe the changes of tumor volume in mice.After the mice were sacrificed,the xenograft tissues were taken for paraffin embedding,sectioning and immunohistochemistry.Immunohistochemistry(IHC)was used to detect the expression of CDK1,GP130 and p-STAT3 in xenograft tissues.Other two group of nude mice,A549 cells were injected subcutaneously into nude mice.After 19 days,the mice administered an intraperitoneal injection of DFO(16 mg/kg)dissolved in 0.9% Na Cl,or saline solution alone,once a day for 12 days.After the mice were sacrificed,the xenograft tissues were taken for paraffin embedding,sectioning,IHC.We detected the expression of GP130 and p-STAT3 in xenograft tissue.In addition,two pairs of lung cancer patients tissue samples(including lung cancer tissue and adjacent normal lung tissues),and tissue microarrays containing 36 pairs of lung cancer tissues and adjacent normal lung tissues,IHC staining for CDK1,GP130,p-STAT3 in lung cancer tissues and adjacent normal lung tissues.Result: 1.The effect of iron,CDK1,STAT3 on the colony formation and sphere formation in lung cancer cells: In human lung adenocarcinoma cells A549 and 1792,the addition of FS significantly increased the number of soft agarose colony formation;in the low-adherent 96-well plates sphere culture assay,the size of sphere was larger in FS treated group.There was a corresponding decrease in sphere size at different concentrations of iron chelator DFO,and the use of CDK1 inhibitors or STAT3 inhibitors significantly inhibited the sphere formation in lung cancer cell lines.Knockdown of either CDK1 or STAT3 blocked ironmediated colony formation.2.The molecular mechanism underlying on the activation of STAT3 signaling by irondependent CDK1 The expression of GP130 and p-STAT3 was increased in A549 and 1792 cells after FS treated,and their expression was significantly decreased after CDK1 i addition.Similarly,si RNA knockdown of CDK1 expression inhibited the expression of GP130 and p-STAT3;si RNA knockdown GP130 reduced p-STAT3 expression,JAK inhibitor significantly inhibited p-STAT3 expression,but had no significant effect on GP130 expression;in the presence of IL-6,STAT3 signaling pathway could still be blocked by CDK1i;different concentrations of iron chelator DFO decreased the expression of GP130 and p-STAT3 at different degrees.The transcriptional regulation of GP130 by iron-dependent CDK1 was excluded by q PCR.The degradation rate of GP130 in A549 cells treated with FS and CDK1 i was not significantly changed after CHX treatment,using MG132 and CHL could not revert to the decrease of GP130 caused by CDK1 inhibition or knockdown;4E1RCat significantly decreased GP130 expression,indicating that GP130 is a cap-dependent translation protein;FS promotes 4E-BP1 phosphorylation,while CDK1 i or si CDK1 decreases p-4E-BP1;in vitro protein phosphorylation assay exhibited a higher level of 4E-BP1 phosphorylation by CDK1/Cyclin B1 in the presence of FS;in vitro protein translation assay showed that the 4E-BP1 group significantly inhibited protein translation.While the CDK1/Cyclin B1 group and CDK1/Cyclin B + FS group restored the ability of protein translation or even had a higher ability of protein translation.3.The role and mechanism of iron-dependent CDK1 in human lung tumor xenograft Knockdown of CDK1 by sh RNA significantly inhibited the growth of xenograft in nude mice,as well as the expression of GP130 and p-STAT3.DFO treatment decreased the growth of xenograft,and the expression of GP130 and p-STAT3.In the tissue sections of lung cancer patients and in the tissue microarray,the expression levels of CDK1,GP130 and p-STAT3 in lung cancer tissues were significantly higher than those in adjacent normal lung tissues.Conclusion: 1.Iron enhances the ability of soft agarose colony formation and sphere formation in lung cancer cells;CDK1 and STAT3 play an indispensable role in the iron-mediated colony formation or sphere formation in lung cancer cells.2.This study was firstly proposed the closely correlation among iron,CDK1 and STAT3 signaling in lung carcinogenesis,iron-dependent CDK1 activates STAT3 signaling pathway.It was firstly proposed that iron-dependent CDK1 promotes protein capdependent translation by phosphorylating 4E-BP1,thereby up-regulating GP130 expression and promoting STAT3 signaling pathway activation.3.Iron-dependent CDK1 plays an essential role in the growth of human lung adenocarcinoma xenograft in nude mice.The CDK1/GP130/STAT3 signaling is significantly higher in lung cancer tissues than in adjacent normal lung tissues.Our results may contribute to the development of potential strategies to target GP130/STAT3 signaling and suppress lung cancer via CDK1 blockade and iron deprivation.