| MPNSTs are an aggressive group of soft tissue sarcomas that can arise sporadically,in the context of NF1,or at a site of prior irradiation.The overall incidence of MPNST in the general population is 1/100000.Although MPNST are rare,but they can become lifethreatening tumors which often metastasize and have a poor prognosis.5-year survival rate of MPNST patients is just 35%–50%,even with aggressive surgery and chemotherapy.Because of its aggressive progression,invasiveness and metastasis,MPNST has a high malignant potential and poor prognosis,with high rates of relapse following multimodality therapy in early disease,low response rates to cytotoxic chemotherapy in advanced disease,and propensity for rapid disease progression and high mortality.In general,standard sarcoma treatment regimens are adapted for chemotherapy for MPNST,including surgical excision with radiation and chemotherapy with agents such as doxorubicin and ifosfamide,although MPNST do not respond well to cytotoxic chemotherapy.To date,there are no effective chemotherapeutic agents for MPNST,which indicates an urgent need for novel targeted therapies.In this study,it's the first time to analyze the drug sensitivity of MPNST through high throughput screen,which helps to find active compounds and drug targets.Based ont the top hits from the screen,we studied the roles and mechanisms of two important kinases,p21 activated kinases(PAKs)and polo like kinase 1(PLK1)in MPNST.Part ? High throughput drug screen of MPNSTWe set up a platform to test MPNST cells against a comprehensive set of small molecular cancer drugs though high throughput screening.We then developed a panel of highly relevant drugs to MPNST that included a comprehensive set of small molecular inhibitors of MEK,RAF,RAS,FTI,PAK,ERK,a representative set of drugs against many other cancer pathways including Wnt,Hedgehog,p53,EGF,HDAC.Meanwhile,the Spearman's rank correlation coefficient was used to compare the consistency.Finally,we pharmacologically induced one MPNST cell line to be resistant to one MEK inhibitor and tested the drug sentivity through the high throughput screen platform we have set up.The results were as followed:(1)We found that MPNST cells were sensitive to a subset of these drugs,including MEK inhibitors,PAK inhibitors,as well as protease inhibitors.In addition,the screen identified some new targets that have not been studied in MPNST before,such as Polo like kinase;Aurora kinase and nicotinamide phosphoribosyl transferase(NAMPT),etc.(2)Our study also seeks to assess the consistency of drug-dose response as a single MPNST cell line was treated with a panel of anticancer drugs.Different viability assays used to measure ATP,oxidation-reduction,and nuclei counts were employed,as well as different drug compound treatment conditions.Finally,our data analysis model to calculate IC50 values was compared to a different analysis model to assess the reliability of our IC50 s.Overall,many factors can contribute to discordance between IC50 values,including varying levels of cytokines in growth media,cell line selection,cell viability assay,drug concentration range,and storage of drug compounds.Based on the results of our study,we argue one of the largest factors of discordance may be the data analysis model used to estimate IC50 values.(3)As a profound study,one MPNST cells were induced to be resistant to MEK inhibitors through drug induce.Drug sensitivity and resitance were also measured by the High throughput screen platform setup above.Through the research above,we performed a large scale drug sensitivity screen via the high throughput screening platform and found the most sensitive drugs,including the Mek inhibitors,some Pak inhibitors,PLK1 inhibitors as well as some proteasome inhibitors.Additionally,we induced one MPNST cell line to be resistant to Mek inhibitor and tested the drug sensitivity by the platform we have set up above.Part ? The role and mechanism of Pak1/2/3 in MPNSTMore than 50% of MPSNTs are associated with mutations in NF1 tumor suppressor gene,resulting in activation of Ras and its effectors,including the Raf/Mek/Erk and PI3K/Akt/m TORC1 signaling cascades,and also the WNT/?-catenin pathway.As Group I p21-activated kinases(Group I Paks,PAK1/2/3)have been shown to modulate Ras-driven oncogenesis,we asked if these enzymes might regulate signaling in MPNST.In this study,we used immunohistochemistry and Western blots to determine the expression of Pak1/2/3 in MPNST sections and cell lines.Through inhibiting Pak1/2/3 by small molecular inhibitors,we studied its role and mechanism MPNST.The results were as followed:(1)Using immunohistochemistry and Western blots,we found the expression level was highly increased in MPNST comparing to normal nerve and neurofibromas indicating there is a a strong positive correlation between the activity of PAK1/2/3 and the stage of human MPNST.(2)We determined that reducing Group I Pak activity diminished MPNST cell proliferation and motility,but that these effects were not accompanied by significant blockade of the Raf/Mek/Erk pathway,but rather by reductions in Akt and ?-catenin activity.(3)Though an array to identify transcription factors and western blots,we found that Pak regulates the expresson of p53 by controling translation of CAP-dependent messages of p53.(4)To synthesize IPA-3 analogs and tested their activities through cell viability assays.Using IPA-3 as a control,Bis(2-naphthyl)disulfide was found to be more active than IPA-3,of which function and mechanism need to be further studied in the future.From the study above,we confirmed that Pak1/2/3 played an important role in MPNST and the expression level of Pak1/2/3 can be used as a marker of the stage of MPNST.Part ? The role and mechanism of polo like kinase 1 in MPNSTPLK1 plays a central role in precisely regulating the cell division and maintaining genome stability in mitosis,spindle assembly,and DNA damage response.Previous studies have shown that PLK1 is highly expressed in most of human cancers,and its overexpression is associated with poor prognosis in cancer patients.Blocking the expression of PLK1 by antibody,RNA interference(RNAi),or kinase inhibitors will effectively inhibit the proliferation of tumor cells and induce apoptosis of tumor cells.So far,there is no available study of PLK1 in MPNST.It's the first time for us to learn how PLK1 expresses and functions in MPNST.In this study,we determined the expression levels of PLK1 from a microarray analysis of NF1 associated MPNSTs.Meanwhile,we study the role and mechanism by MTS assay,wound healing assay and si RNA transfection assay.The results were as followed:(1)The high throughput screen data showed that PLK1 inhibitors had a high sensitivity inhibiting the proliferation of MPNST cells.(2)The microarray data showed that PLK1 is overexpressed in human MPNST.(3)Though MTS assays,wound-healing assays,cell morphological analysis and si RNA screen assays,we identified that PLK1 inhibition reduced MPNST cells growth and cell cycle progression in vitro.(4)The in vivo assay showed that PLK1 inhibitor,BI6727 reduced MPNST xenograft growth,which was synchronized with Aurora kinases inhibitors.To define cellular processes that may contribute to BI6727-induced tumor stasis,we examined cell-cycle markers using IHC on paraffin sections from MPNST xenografts.There was a marked decrease in the number of cells positively labeled for Ki67 in the BI6727-treated tissue as compared with vehicle-treated tissue samples indicating a reduction in actively proliferating cells.No difference was observed in the number of cells labeled for cleaved caspase-3,indicating no effect on apoptosis because of BI6727.No change in percent of cells immunolabeled for phospho-histone-3 in the vehicle versus BI6727-treated samples,indicating possible arrest in the G2-M phase of the cell cycle.Cyclin B1,a marker of G2-M phase,increased in the nucleus and cytoplasm of cells,supporting the idea that the BI6727 inhibitor caused tumor cells to stall in G2-M.Thus,we suggested that PLK1 could be a protent target for cancer therapy. |
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