Study On Simultaneously Targeting DNA Damage Repair Pathway And MTORC1/2 Results In Small Cell Lung Cancer Cells Growth Arrest

Background:It is a dominant feature that the classification of tumors based on cell-of-origin in current studies.Although this classification method has the advantage of locating tumors,the heterogeneity of the tumor and emerges of specific targeted drugs make it difficult to meet current precision medicine and individual treatment.What's more,the traditional method of classification has also changed in recent years: for example,some tumor therapeutic regimens were selected according to the histological type and molecular classification of the tumor.However,there is a still a long way to bridge to the ever-changing targeted therapy of tumor disease.The Pan-Cancer Initiative has released a groundbreaking method to classify tumors based on analysis of genomic and molecular data characterizing 33 different types of cancer from more than 10,000 patients,as well as different genomic profiles and comprehensive cross-cancer analysis to date: to classify tumors based on their cellular and genetic makeup and independent of their anatomic site of origin,which is a new method to meet the treatment strategies of frontier targeted therapy.FDA approved anti-PD1 monoclonal antibody nivolumab for the treatment of melanoma,non-small cell lung cancer,renal cell carcinoma,Hodgkin's lymphoma,head and neck cancer,bladder cancer,colorectal cancer,liver cancer,and stomach cancer in 2017,it's the first anti-tumor drug approved based on a genetic feature of cancers rather than the location of the body where the tumor originated indicating that the new classification of tumors will be a promising method to guide the tumor therapies in future.A large number of studies have shown that the PI3K-AKT-m TOR signaling pathway plays an important role in the development of myeloid and lymphoid cells.The abnormal activation of this pathway is closely related to the occurrence,development and metastasis of hematological tumors.Various studies have shown that targeting the abnormal activation molecule PI3K-AKT-m TOR pathway provides a new strategy for the treatment of hematological tumors.m TOR(Mammalian Target of Rapamycin)is a serine/threonine protein kinase that plays an important role in regulating various aspects of cell proliferation,growth and metastasis,m TOR signaling pathway is aberrantly activated in a variety of tumors The occurrence,development and transfer process are closely related.A number of clinical trials have shown that inhibition of m TOR can delay the progression of multiple tumor diseases and prolong the progression-free survival and overall survival time.It's reported that m TOR signaling pathway is aberrantly activated in clinical specimens and cell lines from leukemia,Hodgkin's lymphoma,non-Hodgkin's lymphoma,multiple myeloma and Fahrenheit macroglobulinemia.m TOR inhibitors have also shown to suppress the proliferation of a variety of hematological malignancies.In addition,combination therapy of m TOR inhibitors and traditional drugs may reduce drug resistance through synergistic or additive effects,increase the drug response rate,and provide new treatment methods for the treatment of these blood diseases.MLN0128 is a novel and selective m TORC1/C2 inhibitor that has the effect of inhibiting the proliferation and survival of various tumor cells.In recent years,multiple studies have shown that MLN0128 can inhibit tumor proliferation with little side effects,indicating it's a prominent anti-tumor drug.The p53 gene is considered the most relevant gene related to tumor diseases.p53 mutation is the most frequent abnormal molecular event in most tumor disease.Although the frequency of p53 mutations in hematologic tumors is lower than that in solid tumors,the mutation rates of p53 in chronic lymphocytic leukemia,acute myeloid leukemia,acute lymphoblastic leukemia,and multiple myeloma are approximately 10-20%,3-8%,3%,and10-12%,respectively,the p53 mutations-induced drug resistance,shorter overall survival times,and worse prognosis have drawn more attention in recently years.the updated NCCN guideline published 2016 considers that p53 mutation is a independent risk factors for acute myeloid leukemia,chronic lymphocytic leukemia,and small lymphocyticlymphoma,suggesting that p53 is an key factor involved in hematologic malignancies.One of the most important functions of P53 is to check whether the DNA is correctly replicated during cell replication.If errors occur during DNA replication,P53 will function to arrest the cell at the G1/S checkpoint during the cell cycle.Only if repaired cells can continue to divide,while those unrepaired cells fell to the apoptotic program.The p53-mutant tumor cells can easily pass the G1/S checkpoint,therefore the G2/M checkpoint becomes an important factor in determining cell fate.Hence,the TP53-mutated tumor cells are more dependent on the repair ability of the G2/M checkpoint.p53 is a key molecule with many downstream key effecotrs,targeting P53 may bring obvious side effects,and the FDA has not approved any targeted drugs for P53,gene therapy for p53 mutation,targeting Cancer vaccines and related anticancer drugs are only in the early stages of clinical trials.The G2/M checkpoint has also emerged a new treatment strategy for the treatment of p53 mutations.The core regulatory molecules of the G2/M checkpoint include:WEE1,Chk1/2,CDC25,etc.WEE1 is an inhibitory protein kinase that regulates cells from the G2 phase to the M phase.Application of WEE1 inhibitor can premature DNA-damaged cells into M phase and induce DNA damage as well as cell death,while normal cells under normal TP53 regulation still waived WEE1 inhibition effect.Several studies have shown that WEE1 inhibitor AZD1775 plays a significant anti-tumor effect in acute myeloid leukemia,acute myeloid leukemia,and diffuse large B-cell lymphoma with mutations in the P53 gene.AZD1775 is an ATP-competitive selective inhibitor of WEE1 kinase that can induce DNA damage,allowing cells with DNA damage to premature into M phase of cell division,eventually leading to cell death.Combination therapy,a treatment modality that combines two or more therapeutic agents,is a cornerstone of cancer therapy.Combination therapy with two drugs or multiple drugs can work on different targets;the synergistic effects bet WEEn two drugs or among the various drugs can enhance the anti-tumor effects and attenuate drug resistance,decrease the growth and potent metastasis,reduce the amount of stem cell and induce tumor cell apoptosis.The project is designed to detect the anti-tumor effect of combinaiton of WEE1 inhibitor AZD177 and m TOR inhibitor MLN0128 in SCLC and explored the underlying mechanisms.Methods Experiments in vitro1.MTT assay and clonogenicity assay were used to detect the anti-proliferation effect of AZD1775?MLN0128 and combiantion treatment in cultured H69 and H82 cells.Synergistic effect was evaluated by the COMBOSYN software proposed by Prof.Ting Chao Chou,Annexin V/PI and Western Blot were applied to monitor cell apoptosis,Brdu-FITC/7-AAD kit was adoptted to test cell proliferation and cell cycle distribution,Western Blot was also used to detect downstream m TOR-Akt pathway and WEE1-Cdc2 activity.2.In order to further explore the mechanisms of AZD1775?MLN0128 and combination theray-induced cell apoptosis,CHOP-sh RNA was transduced into SCLC cells to detect the role of ER stress in AZD1775?MLN0128and combination theray induced cell apoptosis.Experiments in vivo Subcutaneous SCLC xenograft models were established for in vivo studies1.Tumor volumes were measured after AZD1775?MLN0128 and combination therapies were administrated;2.Immunohistochemistry and Western blot were appled to detect the signal pathway after AZD1775?MLN0128 and combination therapies were administrated.Results1.AZD1775?MLN0128 and combination therapy dramatically induced cytotoxicity in cultured SCLC cells,as demonstrated,either AZD1775 or MLN0128 alone had moderate effect on H69 and H82 cancer cell proliferaion in a dose-dependent manner,whereas combination of these two agents induces dramatic growth inhibition and shows synergistic effect bet WEEn the two drugs.Annexin V/PI and western blot shows that AZD1775?MLN0128 and combination therapy can induce SCLC cell apoptosis;Brdu-FITC/7-AAD demonstrates that AZD1775?MLN0128 and combination therapy can induce cell cycle arrest.2.Western Blot results showed that AZD1775?MLN0128 and combination therapy dramatically decreased cell cycle related proteins p-WEE1,p-Cdc2 and Cyclin B1 expression,p-Histone-3 was observed increased suggesting increased premature mitotic entries,increased the level of p-gH2AX indicate persistent unrepaired DNA damage in H69 and H82 cells.MLN0128 treatment mainly diminished phosphorylation of m TOR-Akt pahtway proteins.Administration of AZD1775?MLN0128 or combination therapy attenuated Bcl-2 expression and increased Bim.What't more,Increased GRP78/Bip expression was detected upon single agent treatment and reached to a higher level in combination treatment,indicating an induction of ER stress in SCLC cells.Increased CHOP expression was also evident upon treatment.We have also observed increased p-PERK and p-e IF2? upon treatment.Taking advantage of our well-established lentinvirus mediated sh RNA knockdown assay,we silenced CHOP expression using two sh RNA followed by incubation with both inhibitors to check the role of ER stress in the two inhibitors-induced SCLC cell apoptosis.We have found that AZD1775+MLN0128treatment increased more cell apoptosis in LUC group of H69 and H82 cells,while AZD1775+MLN0128 augmented significantly dereased cell apoptosis in CHOP silenced group in H69 and H82 cells,suggesting that CHOP plays a key role in AZD1775+MLN0128-induced cell apoptosis;3.Significant tumor repression was evident in both H69 and H82 xenograft tumors after administrating of AZD1775?MLN0128 or combination therapies.Particularly,although AZD1775 alone was able to suppress both xenograft tumors,it is less potent than MLN0128.Interestingly,H69 xenograft tumors treated with MLN0128 alone exhibited similar tumor growth effect as H69 xenografts receiving combination therapy.In H82 xenograft tumors,both MLN0128 as a single agent and in combination with AZD1775 significantly suppressed tumor growth;4.By western blot analysis,there was a significant reduction of p-Akt and p-m TOR activity in mice treated with MLN0128 alone and in combination.Significantly increased levels of cl-caspase 3 and cl-PARP were detected in both treated xenograft tumors.Increased premature mitotic entry as evident by increased p-Histone 3,a marker for mitosis was detected in both treated xenograft tumors,especially in tumors treated with combinatorial therapy.Immunohistochemistry using H69 and H82 xenograft tumors have further confirmed suppressed p-m TOR activity,increased mitotic entries,decreased cell proliferation(Ki67 positive cells)and increased cell apoptosis(cl-caspase-3 positive cells)as well as ER stress marker CHOP upon single or combinatorial treatment.These results were very well correlated with tumor regression results obtained with H69 and H82 xenograft mice.Conclusion1.Combination of AZD1775 and MLN0128 synergistially suppress SCLC cells proliferation,which suggesting the potential clinical application value;2.Combination of AZD1775 and MLN0128 can induce SCLC cell apoptosis,er stress plays a key role in mediating AZD1775 and MLN0128-induced cell apoptosis.