The Research Based On Molecular Signatures Of Tumor Radiosensitivity And New Strategy Of Radiotherapy Sensitiz

Part ?:Radiosensitization by a new WNT inhibitor LGK974 and its potential mechanismPurpose:Our goal is to identify a novel molecular targeted radiosensitizer to enhance the therapeutic outcome of cancer radiotherapy.LGK974 is the newest WNT signaling inhibitor currently under clinical trials in the United States and Europe.LGK974 targets Porcupine,a critical WNT signaling component which is frequently upregulated in cancer.WNT signaling has been linked to radiosensitivity,however,how LGK974 might impact tumor cell radio-responsiveness has not been reported.Methods:Cultured tumor cell lines including cervical cancer cell line HeLa(integralMLHl)and colorectal cancer cell line HCT116(MLH1 deficiency),AN3CA(endometrial cancer,MLH1 deficiency),and A2789/cp70(Cisplatin resistance ovarian cancer cell line,MLH1 deficiency)were utilized to assess the radiosensitivity in the presence or absence of LGK974 with a colony formation assay after ionizing radiation(IR).Genetic complementation of the MLH1 gene into HCT116 cells were achieved by transient transfection and validated by Western Blot analysis.We also assessed IR-induced ?-H2A focus formation with immunofluorescence microscopy.Further,we conducted a kinomics analysis to assess the alteration of the kinome,a global description of kinases and kinase signaling in the presence of LGK974 in the isogenic MLH1 background cells.Results:In the initial screen,we found that LGK974 only possessed a radiosensitization effect in HCT116,AN3CA and A2789/cp70 cells which are deficient in MLH1.Reintroducing MLH1 back into HCT116 cells mitigated the radiosensitization effect.In cells treated with LGK974,?-H2A focus formation was significantly reduced in the absence of MLH1.Complementation of MLH1 diminished the LGK974 effect on IR-induced ?-H2A focus formation.Kinomic profiling analysis of pathways affected revealed a decrease of PRKACa and PRKAC? targets in the presence of LGK974.Conclusions:As a novel WNT-signaling pathway inhibitor,LGK974 can induce radiosensitization in the cancer which is absence of MLH-1,a mismatch DNA repair gene.We conclude that the mechanism of action is through inhibition of DNA double strand break repair and impacting PRKACa and PRKAC? phosphorylation targets.These findings have significant clinical impact,as they highlight a novel mechanism of LGK974 that can be used as a powerful radiosensitizer in a subset of cancer patients.Future clinical trials on this direction should follow.Part?:A radiosensitivity MiRNA validated by the TCGA database for squamous cell carcinomas of head and neckPurpose:MicroRNA,a class of small non-coding RNAs,play critical roles in the cellular response to DNA damage induced by ionizing irradiation(IR).Growing evidence shows alteration of miRNAs,in response to radiation,controls cellular radiosensitivity in DNA damage response pathways.However,it is less clear about the clinical relevance of miRNA regulation in radiosensitivity.Methods:Using an in vitro system,we conducted microarray to identify a miRNA signature to assess radiosensitivity.The data were validated by analyzing available Squamous Cell Carcinoma(SCC)samples in the cancer genome atlas(TCGA)database.A total of 28 miRNAs showed differential alteration in response to IR in an Ataxia-Telangiectasia Mutated(ATM)kinase-dependent manner.Results:We validated the list and identified five miRNA signatures that can predict radiation responsiveness in SCC.Furthermore,we found that the expression level of ATM in these patients was correlated with the radiation responsiveness.Conclusions:We demonstrate the feasibility of using a miRNA signature to predict the clinical responsiveness of SCC radiotherapy.Part ?:RAS Promotes Proliferation and Resistances to Apoptosis in MeningiomaPurpose:In this study,we investigated the influence of elevated RAS expression on the growth of meningioma in vivo and in vitro.Methods:The IOMM-LEE cells,representing a cell line derived from malignant meningioma,were divided into blank control group(cells without any drug treatment),negative control group(cells treated with an equal volume of normal saline to replace drug),and farnesyl thiosalicylic acid(FTS)-treated group(cells treated with FTS).Methyl-thiazole-tetrazolium bromide(MTT)assay and flow cytometer were utilized to determine the proliferation and apoptosis,respectively,of IOMM-LEE cells after RAS inhibition.Western blot analysis was used for semi-quantitative analysis of p-ERK and p-AKT levels.Animal model of human meningioma was established with sub-renal capsule transplantation,and mice were divided into two groups:experimental group(50 mg/kg group,75 mg/kg group,and 100 mg/kg,hypodermic injection with FTS)and control group.Proliferating cell nuclear antigen(PCNA)was detected by immunohistochemistry(IHC).Western blot analysis was used for detecting ERK and AKT signal pathway.Results:The proliferation of IOMM-LEE cells decreased dramatically and apoptosis rate increased significantly in FTS-treated group compared to blank control group and negative control group(all P<0.05).At FTS concentration of 75 ?mol/L,the apoptosis rate of IOMM-LEE cells reduced significantly over time(P<0.05).Cell cycle analysis showed that IOMM-LEE cells exhibited G1-arrest in the FTS-treated group,compared to no cell-cycle arrest in blank control group and the negative control group(P<0.05).Further,significantly decreased ERK and AKT phosphorylation levels were detected in IOMM-Lee cells after FTS(75 ?mol/L)treatment for 48 h,compared to blank control group and negative control group(P<0.05).The results in vivo experiments showed that after FTS treatment,tumor volume,PCNA LI,and the levels of p-ERK and p-Akt decreased significantly in 75 mg/kg group and 100 mg/kg group when compared with the control group and 50 mg/kg group(all P<0.05).Conclusions:Our findings provide strong evidence that RAS protein is highly expressed in meningioma cells,and the RAS activity is inhibited by downregulating ERK and AKT signal pathway,which may further inhibit the growth of meningioma.