| Esophageal Cancer(EC) is the fourth cause of cancer associated death and has high death and poor prognosis in China. Though several treatments of EC which include surgery, radiotherapy, chemotherapy and combination with them have been improved, the effect is still limited. Even more, the overall 5-year survival of EC is about 17% and is lower than the median 5-year survival of all tumors in China.However, the carcinogesis mechanism of EC is not clear. So it is important to further research on the carcinogesis mechanism of EC for understanding the pathogenesis of EC and to provide theoretical basis for making effective treatments and lowering the mortality and incidence of EC. Esophageal Squamous Cell Carcinoma(ESCC)accounts for the major type of esophageal cancer and it is confirms that abnormal signal pathways are associated with the development of ESCC. Therefore, screening new therapeutic targets and making effective treatments are one of the ESCC clinical problems.Cancer genome landscapes found that 79 key genes, which promoted the development of ESCC and were involved in 12 signal transduction pathways, had the function in genome stability, cell survival and cell fate in ESCC. Recent researches confirmed that aberrant activation of signal pathways, such as AKT and RAS,promoted the development of ESCC and were involved in the proliferation,differentiation, and other processes of ESCC. These studies provide theoretical support to illuminate the function of the signal pathways, which would help to screen key signaling pathway targets and inhibitors, to block the development of ESCC, to make effective treatments and improve the ESCC patients’ survival rate.PDZ binding-kinase/T-LAK cell-originated Protein kinase(PBK/TOPK) is a newly discovered serine-threonine kinase which belongs to the MAPKK family and is a branch of MEK1/2 and MKK7 from phylogenetic tree. TOPK participates in a variety of biological processes, such as tumor development, cell proliferation,apoptosis, cell cycle, and even inflammation. Activation of TOPK is an important step in early mitosis process. TOPK and cdk1/cyclin B1 complex could promote cell division through phosphorylating protein regulator of cytokinesis1 and then accelates G2/M phase to prompt cell proliferation. TOPK kinase can bind with DCD structure domain of tumor suppressor gene p53, which could block the role of p53 and promote the expression of cell cycle proteins such as p21. Recent study showed that Cdk1-actived TOPK kinase instantaneously controlled a variety of DNA connection proteins in cell division, even more regulated the precursors of these proteins in a few minutes. C2H2 zinc finger protein is the categories of conservative protein which contains more conservative area of zinc finger domain structure. TOPK kinase is reported as the first kinase which regulates and controls the whole transcription factor protein kinase by binding with their conservative domain in mitosis. It is confirmed that TOPK is high expression in many kinds of malignant tumors, such as leukemia,lymphoma and breast cancer, skin cancer, cervical cancer, lung cancer and colon cancer and so on, and is closely related to the prognosis. TOPK is involved in tumor development and metastasis by activating different signaling pathways in different tumors. Further more, TOPK is not only involved in cancer development, but also correlated with cancer drug resistance. Breast cancer patients, resistant to pury ketone and methyl hydroxy valeric acid treatment, have high expression of TOPK induced by YAP which maybe play an important role in promoting breast cancer cell proliferation. Latest literatures reported that EGFR inhibitors resistance in lung cancer cells could be promoted through TOPK kinase directly phosphorylated c-Jun at S73 and S63. Therefore, these studies have shown that TOPK has the potential to be an effective therapeutic target in cancer treatments.However,there are no reports about the role and mechanism of TOPK in ESCC.Therefore, we focus on the role and mechanism of TOPK in ESCC, and it provides theoretical evidences for TOPK as new target in targetic therapy of ESCC. In this study, we first detected TOPK expression in esophageal cancer through the esophageal tissue micro array, and evaluated TOPK relevance to pathological data and prognosis of esophageal cancer patients. Next, we evaluated the TOPK effect on esophageal cancer cell proliferation by sh TOPK and TOPK inhibitors HI-TOPK-032.We also explored the change of the signal transduction pathways through kinase assay, and found that Y box protein1(YB1) was the phosphorylation target of TOPK by plasma resonance technology(SPR) and mass spectrometry(MS) and identified that TOPK phosphorylated YB1 was at Ser209 and Thr89.The role of YB1 was evaluated in ESCC cell through si RNA. Furthermore, we evaluated the role of TOPK in nude mice xenograft model and our established ESCC patient-derived xenograft(PDX) models. These provided theoretical basis for TOPK as a new target in targetic therapy of ESCC.Part I Expression of TOPK and evaluation of its clinical relevance in ESCCMethods1 A human esophageal squamous carcinoma tissue micro array was used to detect the expression level of TOPK in 100 ESCC patients compared with normal esophageal tissues adjacent to carcinoma2 Analysis of the relationship among TOPK with the clinicopathological characteristics(which included gender, age, differentiation, lymph node metastasis and tumor infiltrating) and the prognosis of ESCC patientsResults1 TOPK is highly expressed in human ESCC tissues compared with normal esophageal tissues adjacent to carcinoma The positive expression of TOPK was mainly in the cytoplasm and/or cell nucleus with different intensity brown staining. According to score values, low expression group which was 34 cases(score≤3) and high expression which was 66cases(score > 3) were divided in all the ESCC patients and the expression was negative in normal tissue adjacent to carcinoma.2 Correlational analysis between TOPK with the clinic pathological characteristics in ESCC patients Statistical analysis of the relationship between TOPK expression and the clinical data(age, gender, grade, lymph node metastasis and tumor infiltrating) in the ESCC cases, the results showed that in no lymph node metastasis group, lower expression of TOPK was 21 cases which accounted for 43.48%, high expression was 25 cases which accounted for 56.52%; In lymph node metastasis group, low expression of TOPK was 13 cases which accounted for 25.93%, high expression was 41 cases which accounted for 74.07%, the difference was statistically significant(X2 =5.154, P=0.023).However, it was no correlation between TOPK expression and other clinical data(age, sex, tumor differentiation, tumor infiltrating)(P>0.05).3 Overexpression of TOPK suggests poor prognosis in ESCC patients In the 100 patients, the survival time was 49.75±5.89 months in 34 low TOPK expression patients, 19.82±2.03 months in 66 high TOPK patients. The difference in survival time between low expression and high expression was significant, P < 0.001.Part II Downregulation TOPK inhibits the proliferation of ESCC cells and its molecular mechanismChapter 1 Inhibitory effect of downregulated TOPK on the proliferation in EC9706 and TE13 ESCC cell linesMethods1 Western Blotting to detect the level of TOPK expression in ESCC cell lines2 Knocking down TOPK in EC9706 and TE13 cells by lent virus transfection,Western blotting to detect the knock down efficiency of TOPK in sh TOPK cell lines3 CCK8 assay and soft agar assay to determine the inhibited role of TOPK in the proliferation and colony formation of EC9706 and TE13 sh TOPK cell lines4 CCK8 assay and soft agar assay to evaluate the inhibital effectiveness of TOPK inhibitors HI-TOPK-032 for the proliferation and clony formation of EC9706 and TE13 cell linesResults1 TOPK expression in esophageal squamous cancer cells and establishing the EC9706 and TE13 sh TOPK cell line The expression of TOPK was high in TE1, TE13, EC9706 and EC1 cell lines.We established stable TOPK low expression EC9706 sh TOPK#1 and EC9706 sh TOPK#2, TE13 sh TOPK#1 and TE13 sh TOPK#2 cell lines by knocking down TOPK.2 Knocking down TOPK inhibited the proliferation and clony formation of EC9706 and TE13 cell lines Compared with control group, CCK8 assay and soft agar assay results indicated that the proliferation and clony formation of EC9706 and TE13 were inhibited by knocking down TOPK.3 HI-TOPK-032 inhibited the proliferation and clony formation of EC9706 and TE13 cells CCK8 assay and soft agar assay results showed that HI-TOPK-032 obviously inhibited the proliferation and colony formation of EC9706 and TE13 cells compared with control group.Chapter 2 Downregulated TOPK inhibits the activation of signaling pathway in the ESCC cell linesMethods1 Human Phospho-Kinase Array to detect the changes of signaling pathways after TOPK knockdown in EC9706 cells2 Western blotting to detect the changes of signaling pathways after knocking down TOPK in EC9706 and TE13 cells3 Western blotting to detect the changes of signaling pathways after HI-TOPK-032 treated EC9706 and TE13 cells for 24hResults1 The changes of signaling pathways in EC9706 sh TOPK cell Compared with control sh Mock group, results indicated that the level of Akt1/2/3 S473 was dropped to 0.33 and 0.49 in EC9706 sh TOPK # 1 and EC9706 sh TOPK # 2 respectively, Akt 1/2/3 T308 dropped to 0.85 and 0.51,p70S6 kinase T421 / S424 dropped to 0.57 and 0.23 respectively; ERK1/2 T202 /Y204 dropped to 0.81 and 0.79 respectively.2 Western blotting to assess the results of human phospho-kinase array Knocking down of TOPK and HI-TOPK-032 both inhibited AKT/m TOR/p70S6 K and Erk singal pathway in EC9706 and TE13 cells. These results were consistent with the ones of human phospho-kinase array.Chapter 3 Study on the downstream molecular target of TOPKMethods1 Screening the targets of TOPK kinase in EC9706 cells by surface plasmon resonance(SPR) and mass spectrum(MS).Y box binding protein 1(YBX1, also called YB1) was used in the next research by analysis and validation of the MS results2 Immunohistochemistry to observe TOPK and YB1 expresion in esophageal squamous carcinoma tissues, immunofluorescence analysis to observe TOPK and YB1 expresion in EC9706 and TE13 cells3 Co-Immunoprecipitation of TOPK and YB1 in vitro and in vivo4 In vitro kinase assay to confirm TOPK kinase phosphorylates YB1, MS to detect phosphorylation sites at which TOPK kinase phosphorylates YB1.Results1 Screening the proteins combined with TOPK kinase in EC9706 cells by SPR and MS analysis Proteins whose score was more than 25 were selected from MS result. We focused on analysis 32 proteins which were combined with TOPK kinase. We finally confirmed YB1 as the TOPK kinase protein target through literature analysis and supposed that TOPK kinase phosphorylated YB1 to active the downstream signal pathway.2 TOPK and YB1 were colocalized in ESCC tissues and cells Immunohistochemical results showed YB1 and TOPK appeared together in the cytoplasm of esophageal cancer tissues, immunofluorescence results indicated TOPK and YB1 were con-focused in the cytoplasm of EC9706 and TE13 cells.3 TOPK could bind with YB1 in vitro and in vivo Co-immunoprecipitation confirmed that exogenous TOPK binded with YB1 in HEK293 cells and endogenous TOPK binded with YB1 in EC9706 cells.4 TOPK phosphorylated YB1 at Thr89 and Ser209 in vitro TOPK kinase phosphorylated YB1 by in vitro kinase assay; TOPK kinase phosphorylated YB1 at Thr89 and Ser209 in vitro by MS analysis.Chapter 4 The role of YB1 in the proliferation of EC9706 and TE13 cell linesMethods1 Western blotting to detect the level of YB1 expression in ESCC cell lines2 CCK8 assay and soft agar assay to determine the change of the proliferation and clony formation in EC9706 and TE13 cell lines by si YB13 Western blotting to detect the level of TOPK, p-TOPK and other singal pathway by si YB1Result1 YB1 expression in esophageal cancer cell linesIn order to evaluate YB1 expression in esophageal cancer, we examined the level YB1 in a number of esophageal cancer cell lines, results showed the expression of YB1 was varied in multiple esophageal cancer cell lines. These results indecated that overexpression of YB1 was associated with biological characteristics of esophageal cancer cells.2 Decreasing YB1 inhibited the proliferation of colony formation in EC9706 and TE13 cells The level of YB1 in EC9706 and TE13 cells was apparently decreased by si RNA, CCK8 and soft agar assay showed si YB1 could inhibit the proliferation and colony formation in EC9706 and TE13 cells.3 si YB1 had no effect on p-TOPK and TOPK and downregulated AKT/m TOR/p70S6 K signal pathway in EC9706 and TE13 Western blotting results confirmed that si YB1 had no effect on the expression of p-TOPK and TOPK, inhibited the activation of AKT/m TOR/p70S6 K signaling pathway, and had little influence in ERK pathway.Part III Decreasing TOPK suppresses the growth of xenografts in EC9706 mouse model and ESCC PDX modelChapter 1 Knocking down TOPK suppresses tumor growth of EC9706 cells in a xenograft mouse modelMethods1 Establish an athymic nude xenograft mouse model of EC9706 sh Mock and EC9706 sh TOPK#1 and EC9706 sh TOPK#2 cells, determine the effect of TOPK on the growth in vivo by measuring the tumor size, body weight, diet in mice.2 Immunohistochemical analysis to evaluate TOPK, p-TOPK, Ki67 and downstream signal pathways by knocking down TOPK in an athymic nude xenograft mouse model.Results1 Tumor growth and body weight in mice Measured the tumor volume from the 7th days after injecting cells in subcutaneous, tumor volume was significant difference from 13 days; there was no difference in weight, eating and drinking of mice during the experiment.2 Tumor weight and volume when the mice were sacrificed Mice were sacrificed on day 21, measuring tumor volume and weighing tumor,the results found tumor volume and weight of EC9706 sh TOPK#1 and sh TOPK#2group were reduced compared with control group(P<0.001).3 Activation of signal pathways by knockdown TOPK in exnografts Compared with control group, immunohistochemical results showed p-TOPK,Ki67 and phosphorylation of AKT, m TOR and p70S6 K were decreased in exnografts of EC9706 sh TOPK#1 andsh TOPK#2(P<0.05).Chapter 2 Establish human ECSS patient-derived exnograft modelsMethods1 Implant subcutaneously 26 cases of ESCC tissues into severe combined immunodeficiency(SCID) mice, grow and passage2 Tumor growth curve, HE and CK5/6, p40, p60 immunohistochemical analysis to evaluate established PDX modelsResults1 Clinical data The success rate was 53.8% and 14 ESCC PDX models were established. In 26 cases, 17 were male, 9 were women, age was from 46 to 82, 17 were stage II people,3 were phase III, 2 were II-III, 3 were I phase, and 1 was I-II phase.2 Tumor growth curve, pathological evaluation and immunohistochemical analysis in established PDX models EG2, EG3 and EG5 were represented as 14 established ESCC PDX xenograft model. Their growth curve indicated the third passage was shorter than the first and second passage; HE and CK5/6, p40, p63 immunohistochemistry showed that pathological type and positive expression of CK5/6, p40, p63 in PDX models were consistent with patient tumor tissue.Chapter 3 HI-TOPK-032 suppresses tumor growth in ESCC PDX model Methods1 Western blotting to select one p-TOPK positive(EG5) model and one negative(EG2) model from established ESCC PDX models; To determine the chemo therapeutic effect of HI-TOPK-032 in EG5 and EG2 PDX models from tumor volume, tumor weigh, mice weight, diet and drink.2 Immunohistochemical analysis to evaluate the expression level of phosphorylated TOPK, AKT, m TOR and p70S6 K and Ki-67 in HI-TOPK-032 treated EG5 and EG2 PDX models.Results1 Clincal data of patient PDX of p-TOPK positive case(EG5) and p-TOPK negative case(EG2) were treated with HI-TOPK-032. Both of cases were from the first affiliated hospital of Zheng Zhou university, they were both ESCC and had no chemothraphy and radiotherapy before surgery. EG2 and EG5 patients were both men, age was 64 and61 respectively, differentiation was middle-high and differentiation respectively.They both were in stage IIa(T2N0M0) and had no lymph metastasis.2 Tumor growth and body weight in mice during HI-TOPK-032 treatment Tumor volume in HI-TOPK-032 treated group was smaller than in control group in EG5 PDX model from 12 th day, P=0.002. However, there was no difference between HI-TOPK-032 treated group and control group in EG2 PDX model during all treatment, P> 0.05. Furthermore, there were no difference in mice weight, diet and drink between HI-TOPK-032 treated group and control group in EG5 and EG2 PDX models during the experiment.3 Tumor weight and volume when the mice were sacrificed Mice were sacrificed on day 29, measuring tumor volume and weighting tumor,the results showed tumor volume(P=0.001) and weight(P=0.004) of HI-TOPK-032 treated group were reduced compared with control group in EG5 PDX model,however, there was no difference in EG2 PDX model(P>0.05).4 Inhibition of signal pathways by HI-TOPK-032 treatment in PDX models Compared with control group, immunohistochemical results showed p-TOPK,Ki67 and phosphorylation levels of AKT, m TOR and p70S6 K were decreased in exnografts of HI-TOPK-032 treated group in EG5 PDX model(P<0.05). However,there was no difference in these indexes between HI-TOPK-032 treated group and control group in EG2 PDX model(P>0.05).Conclusions1 TOPK is highly expressed in ESCC tissues compared with normal esophageal tissues adjacent to carcinoma. TOPK is closely relatived with lymph node metastasis,and has a negative correlation with survival time. However, TOPK has no correlation with other clinical indexes(gender, age, differentiation and tumor infiltrating).2 Downregulated the expression of TOPK expression can inhibit the proliferation and colony formation of ESCC cells and the growth of ESCC exnografts.These results indicate that TOPK play an important role in the growth of esophageal squamous cell cancer. TOPK has the potency to be an effective molecular target for prevention and treatment in ESCC.3 Decreasing the expression of TOPK can inhibit activating of AKT/m TOR/p70S6 K and ERK signaling pathway. TOPK phosphorylates YB1 at Thr89 and Ser209. si YB1 can inhibit the proliferation and activation of AKT/m TOR/p70S6 K signal pathways in ESCC cells. These results indecate that one of mechanism which TOPK prompts the growth of ESCC is that TOPK phosphorylates YB1 and then actives AKT/m TOR/p70S6 K signal pathways.4 We successfully established ESCC patient derived exnograft(PDX) models.PDX models provide a preclinical platform for further understanding the molecular mechanisms of ESCC, screening effective therapeutic targets and evaluating new therapeutic strategies for ESCC patients. |
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