Study On KRAS Allele Specific Anticancer Effect And Mechanisms Of Alisertib,An Aurora Kinase A Inhibitor In Colorectal Cancer

RAS mutations occur in～20-30%of malignant tumors overall,and notably in-45%of colorectal cancers(CRCs).RAS mutations are essential for tumor initiation and maintenance.Moreover,RAS mutations are often associated with a poor prognosis.KRAS G12C,G12D,G12V,G12V,13D,and BRAF V600E mutations are often found in CRC,contributing to tumorigenesis and progression.On the other hand,KRAS is subject to multiple tiers regulation,including kinase.Aurora kinases has been implicated in many types of tumor onsets and progession,making them as a promising therapeutic targets.Alisertib(ALS),selectively inhibits Aurora kinase A(AURKA)and has shown potent anticancer activity in vivo and in vitro studies,but the latent anticancer effect of ALS on CRC remains unclear in the context of different KRAS mutations.Objective1、To explore the effects of ALS on the RAS signaling pathway in CRC liens in the context of different KRAS mutations.2、To explore the molecular targets and proteomic response of ALS in colorectal cancer HT29 and Caco-2 cell lines.3、To detect the effect on RAS pathways and programined cell death and reveal the molecular mechanism of ALS on HT29 and Caco-2 cells.4、To detect the anticancer effect of ALS in addition to MEK inhibitor on different KRAS alleles expressing CRC lines.MethodsThis study aimed to evaluate the effect of ALS on RAS signaling pathway in a panel of CRC lines expressing different KRAS alleles,including Caco-2(KRAS WT),Colo-678(KRAS G12D),SK-CO-1(KRAS G12V),HCT116(KRAS G13D),CCCL-18(KRAS A146T)and HT29(BRAF V600E).We examined the effect of ALS on the level of RAS-GTP using pulldwon.To determine whether ALS effectively affect RAS signaling pathway in these cell lines,we examined the signal ouput in PI3K/Akt and MAPK pathways by Western Blotting method.We next assessed the effects of ALS on cell apoptosis and cell autophagy of the CRC lines by testing cleaved PARP,LC3-Ⅱ,and LC3-I.We examined the effect of ALS on the viability of HT29 and Caco-2 cells using MTT assay and the values of IC50 were determined from the relative viability over ALS concentration curve.To determine whether ALS effectively targeted AURKA in HT29 and Caco-2 cells,we examined the expression level of total AURKA and phosphorylation level of AURKA by Wester Blotting method.We performed a stable isotope labeling by amino acids in cell culture(SILAC)-based proteomic study to quantitatively detect the molecular interactome in HT-29 and Caco-2 cells responding to ALS.We next assessed the effects of different concentrations and times of ALS on the cell cycle distribution,cell apoptosis and cell autophagy of HT29 and Caco-2 cells by flow cytometry.To further examine the cellular autophagy level,confocal microscopic examination was carried out.Western blotting methods were used to determine the expression level of key regulators involved in cell cycle(CDK1/CDC2,p-CDC2(Tyr15),cyclin B1,p-cyclin B1(Ser133),p-CDC25C(Ser216),PLK1,p53 and p21/Wafl),apoptosis(Bcl-xl,Bcl-2,Bax,PUMA,cytochrome c,cleaved caspase 3,cleaved caspase 9,cleaved PARP,RIP,p-FADD and FADD),autophagy(p-PI3K,PI3K,p-AMPK,AMPK,p-Akt,Akt,p-p38 MAPK,p38 MAPK,p-mTOR,mTOR,LC3-Ⅱ,LC3-Ⅰ,beclin 1 and PTEN),and EMT(E-cadhcrin,N-cadherin,Slug,TCF-8/ZEB1 and ZO-1)processes.Results1、ALS modulated the active form of KRAS in a RAS allele specific manner across the panel of CRC lines.ALS upregulated RAS-GTP level in Caco-2 cells expressing wildtype KRAS,but did not affect RAS-GTP level in Colo-678 expressing KRAS G12D mutant.ALS also increased RAS-GTP level in SK-CO-1 cells expressing KRAS G12V mutant,but did not influenced RAS-GTP level in CCCL-18 cells expressing KRAS A146T mutant.Moreover,ALS upregulated RAS-GTP level in HCT116 cells expressing KRAS G13D mutant,whereas decreased RAS-GTP level in HT29 cells harboring BRAF V600E mutation.2 s ALS differentiated regulated RAS signaling pathway.ALS inhihited PI3K/Akt signals but activated ERK in Caco-2 cells.ALS did not affected PI3K/Akt and MAPK signa output in Colo-678 cells.ALS attenuated PI3K/Akt and ERK signals in SK-CO-1 cells.ALS suppressed PI3K/Akt and ERK activation in HCT116 cells.ALS increased PI3K/Akt and MAPK signal output in CCCL-18 cells.ALS inhibited PI3K/Akt and ERK activation in HT29 cells.3、ALS induced apoptosis and autophagy in a RAS allele specific manner.ALS induced apoptosis and autophagy in Caco-2 and HT29 cells.ALS did not affect the level of LC3 in colo-678 cells.ALS increased the level of cleaved-PARP and the ratio of LC3-Ⅱ/Ⅰ in SK-CO-Ⅰ cells.ALS increased the level of cleaved-PARP but did not affect the ratio of LC3-Ⅱ/Ⅰ in HCT116 cells.ALS increased the level of cleaved-PARP and the ratio of LC3-Ⅱ/Ⅰ in CCCL-18 cells.4、ALS significantly inhibited the cell proliferation of HT29 and Caco-2 cells.ALS effectively targeted and inhibited AURKA.There were 893 protein molecules identified as the potential targets of ALS in HT-29 cells.The expression level of 459 protein molecules was increased,while the expression level of 434 protein molecules were decreased in HT-29 cells.In Caco-2 cells,There were 582 protein molecules identified as the potential targets of ALS.ALS increased the expression level of 207 proteins,but decreased the expression level of 375 proteins.These protein molecules were subjected to IPA pathway and network analysis.There were 24 and 25 networks of signaling pathway and cellular function which were potentially regulated by ALS in HT-29 and Caco-2 cells,respectively.These included a series of molecules involved in cell proliferation,cell migration,cell invasion,cell metabolism,cell survival,and cell death processes.5、ALS induced cell apoptosis in HT29 and Caco-2 cells in concentiration-andtime-dependent manners.ALS increased the expression level of Bax,PUMA,cytochrome c,cleaved caspase 3,cleaved caspase 9,cleaved PARP and decreased the expression level of Bcl-xl and Bcl-2 in HT29 cells.For Caco-2 cells,ALS increased the expression level of Bcl-2,RIP,p-FADD,cytochrome c,cleaved caspase 3,cleaved caspase 9 and cleaved PARP and decreased the expression level of Bax and FADD and the value of p-FADD/FADD.6、Incubation of HT29 and Caco-2 cells with different concentrations and timesof ALS resulted in a remarkable increase in the percentage of autophagic signal.ALS decreased the expression level of p-PI3K,p-Akt,p-mTOR and the values of p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR,while increasing the expression level of PTEN and beclin 1,and the values of p-AMPK/AMPK,p-p38/p38 and LC3-II/LC3-I in HT29 cells.For Caco-2 cells,ALS decreased the expression level of p-PI3K,p-Akt,p-mTOR and the values of p-PI3K/PI3K and p-p38/p38,while increasing the expression level of PTEN,beclin 1 and LC3-Ⅱ,and the values of p-AMPK/AMPK and LC3-Ⅱ/LC3-Ⅰ.7、In combination of ALS and MEK inhibitor,Selumetinib,enhanced ALSregulatory effects in CRC lines in a RAS allele specific manner.Conclusion1、ALS differentiated regulated RAS signaling pathway and manipulated cellapoptosis and autophagy in RAS allele specific manner.2、ALS effectively targets AURKA and potently inhibits the proliferation of ALSin HT29 and Caco-2 cells.3、ALS induces apoptotic death of HT29 and Caco-2 cells through mitochondrial pathway and death receptor signaling pathway respectively.4、Inhibition of the PI3K/Akt/mTOR pathway and activation of AMPK contribute to the autophagy-inducing effect of ALS on HT29 and Caco-2 cells.There is a differential modulating effect of ALS on p38 MAPK signaling pathway with up-regulation of p38 MAPK signaling in HT29 cells and down-regulation of p38 MAPK signaling in Caco-2 cells.There is a crosstalk between ALS-induced autophagy and apoptosis in HT29 and Caco-2 cells.5、ALS in addition to MEK inhibitor enhanced the anticancer effects of ALS in CRC lines in a RAS allele specific manner,which may represent a new therapeutic strategay for precision therapy of CRC in a RAS allele manner.