突变体能够通过内源性的促进胰腺癌的转移课件.ppt

1、突变体能够通过内源性的促进胰腺癌的转移（优选）突变体能够通过内源性的促进胰腺癌的转移Backgroundprevent senescencePromotemetastasismutant p53drug resistanceInduceapoptosisp53antineoplasticpreventangiogenesisBackground75%p53 mutant highly metastatic poor prognosisdrug resistanceBackgroundKPC cell:stablely express small haipin RNA,lost remaning

2、 p53 wide-type alleKPflC cell:a p53 null cell line KPC mice:develop highly metastatic pancreatic cancer that faithfully mimics the human diseaseIdeasMutant p53 maintain metastatic phenotypethe relationship between Mutant p53 and PDGFRb on metastasisPDGFRb madiates metastasis when Mutant p53 was depl

3、etedimatinib can inhibit metastasis by targeting PDGFRbPDGFRb correlates with disease-free survival PDGFRb as a Downstream Mediator of Mutantp53Methods1.Wound Healing and Invasion Assays2.RNA Sequencing and Data Analysis3.Immunostaining and Microscopy4.qRT-PCR5.PDGFRb Luciferase Reporter Assay6.Coim

4、munoprecipitation and Chromatin Immunoprecipitation7.Immunohistochemistry and Immunofluorescence8.Mouse StudiesQuestionwhether mutant p53 is needed to sustain the metastatic phenotype and how it is regulated？RESULTSKPC+sh.Ctrl cell express mutant p53划痕愈合率划痕愈合率侵袭细胞数侵袭细胞数The invasiveness depend on mut

5、ant p53.Result 1：Sustained Expression of Mutant p53 Is Required for the Invasive Phenotype of Pancreatic Cancer Cellsthe relationship between Mutant p53 and PDGFRb on metastasisResult 5：Modulation of PDGFRb Expression Levels Mediates the Phenotypic Effects of Mutant p53 Depletion In Vivothe relation

6、ship between Mutant p53 and PDGFRb on metastasisPDGFRb as a Downstream Mediator of Mutantp53how mutant p53 mediates the invasive phenotype of PDAC?Ctrl cell express mutant p53p53/PDGFRb敲除Wound Healing and Invasion Assaysdrug resistancePDGFRb Luciferase Reporter Assayhypothesis：PDGFRb could affect ce

7、ll invasion（优选）突变体能够通过内源性的促进胰腺癌的转移Wound Healing and Invasion Assaysmutant p53 promotes invasion,inpart,depends on its ability to enhance PDGFRb expression through the disruption of the inhibitory p73/NF-Y complexwhether PDGFRb levels correlate with the status of p53?抑制细胞增殖所需IC50转移瘤数量转移瘤数量whether mut

8、ant p53 expression was required to sustain the metastatic potential of KPC cells?转移瘤荧光体视成转移瘤荧光体视成像像Summary 1these results demonstrate that mutant p53 can contribute to PDAC invasion and metastasis and that inhibiting its activity can have an antimetastatic effectQuestionhow mutant p53 mediates the i

9、nvasive phenotype of PDAC?mutant p53 can affect the invasive phenotype of pancreatic cancer cells RNA 测序，基因变化测序，基因变化IPA分析分析Result 2：Transcriptional Profiling and Functional Screening Identify PDGFRb as a Downstream Mediator of Mutant p53 in Murine Pancreatic Cancer1：SLC40A12：SNED13：PDGFRbhypothesis：

10、PDGFRb could affect cell invasionPDGFRb转录水平转录水平蛋白表达蛋白表达mutant p53敲除敲除细胞增殖细胞增殖不同亚型对侵袭的影响不同亚型对侵袭的影响不同亚型蛋白表达情况不同亚型蛋白表达情况p73 can repress the transcription of PDGFRbincreased PDGFRb did not confer a selective advantage to tumor cell proliferation抑制细胞增殖所需IC50不同种类肿瘤中Mutp53敲除PDGFRb as a Downstream Mediator

11、of Mutantp53the high disease burden in the pancreas was the primary cause of deathKPflC 细胞侵袭能力mutant p53 can affect p73/NF-YBKPC cell:stablely express small haipin RNA,lost remaning p53 wide-type alleby inhibiting the kinase activity of PDGFRb,imatinib significantly diminishes the metastatic potenti

12、al of pancreatic cancer cells.how mutant p53 mediates the invasive phenotype of PDAC?（优选）突变体能够通过内源性的促进胰腺癌的转移mutant p53 promotes invasion,inpart,depends on its ability to enhance PDGFRb expression through the disruption of the inhibitory p73/NF-Y complex突变体能够通过内源性的促进胰腺癌的转移抑制细胞增殖所需IC50（优选）突变体能够通过内源性的促

13、进胰腺癌的转移the high disease burden in the pancreas was the primary cause of deathPDGFRb as a Downstream Mediator of Mutantp53angiogenesis红色荧光蛋白红色荧光蛋白绿色荧光蛋白绿色荧光蛋白两种细胞比值两种细胞比值increased PDGFRb did not confer a selective advantage to tumor cell proliferation Summary 2 PDGFRb is not required for the prolifer

14、ation and tumorigenic potential of p53 mutant murine cancer cells but specifically impacts their invasive potential.QuestionIf the mutant p53-PDGFRb signaling axis acts in human cancer cells？PDGFRb mRNA 水平水平四种人胰腺癌细胞（四种人胰腺癌细胞（Mutp53敲除）敲除）不同种类肿瘤中不同种类肿瘤中Mutp53敲除敲除Result 3：PDGFRb Mediates Mutant p53 Act

15、ion in Human Cancer Cellsmutp53/PDGFRb 分别敲除，分别敲除，A2.1细胞侵袭情况细胞侵袭情况p53/PDGFRb敲除敲除p53-/-细胞过表达细胞过表达PDGFRbinvasionp53-/-人胰腺癌细胞人胰腺癌细胞Summary 3upregulation of the PDGFRb is important for the action of mutant p53 in PDAC and possibly other tumor types.p73 can repress the transcription of PDGFRbour KPC cells

16、 expressed p73,but not p63 whether the physical interaction of mutant p53 with p73 might impair the ability of p73 to negatively regulate the expression of PDGFRb?mutant p53 can inhibit p73 and p63QuestionKPflC PDGFRb荧光素酶报告基因荧光素酶报告基因免疫共沉淀免疫共沉淀Result 4：Mutant p53 Disrupts the p73/NF-Y Complex to Medi

17、ate PDGFRb Expression and Tumor Cell Invasionby inhibiting the kinase activity of PDGFRb,imatinib significantly diminishes the metastatic potential of pancreatic cancer cells.whether PDGFRb levels correlate with the status of p53?突变体能够通过内源性的促进胰腺癌的转移Wound Healing and Invasion Assaysantineoplasticmuta

18、nt p53 can affect p73/NF-YBimatinib can inhibit metastasis by targeting PDGFRbCrenolanib两种细胞IC50（优选）突变体能够通过内源性的促进胰腺癌的转移Wound Healing and Invasion Assaysthe high disease burden in the pancreas was the primary cause of deathincreased PDGFRb did not confer a selective advantage to tumor cell proliferat

19、ionp73 can repress the transcription of PDGFRbCoimmunoprecipitation and Chromatin Immunoprecipitationabrogation of autocrine activation signaling of PDGFRb leads to a significant reduction of invasion and metastasis driven by mutant p53.突变体能够通过内源性的促进胰腺癌的转移抑制细胞增殖所需IC50p53-/-人胰腺癌细胞Result 7：PDGFRb Expr

20、ession Correlates with Disease-free Survival in Human Pancreatic,Colorectal,and Ovarian Cancer Patients不同种类肿瘤中Mutp53敲除drug resistance how p73 represses PDGFRb transcription?Questionp73NF-YBPDGFRb序列NF-Y binding to the PDGFRB promoter p73/NF-Y interaction hampers NF-Y to bind and activate the PDGFRB p

21、romoter p73 binding with NF-YBmutant p53 can affect p73/NF-YBPDGFRb结合定量结合定量NF-YBNF-YANF-YCwhether the repressive action of p73 on PDGFRb transcription was mediated by NF-Y and modulated by mutant p53?QuestionKPflC 细胞侵袭能细胞侵袭能力力PDGFRb荧光素酶报告基因荧光素酶报告基因KPflC细胞细胞KPC侵袭能力修复侵袭能力修复Summary 4mutant p53 promotes

22、 invasion,inpart,depends on its ability to enhance PDGFRb expression through the disruption of the inhibitory p73/NF-Y complexwhether PDGFRb levels regulate metastatic behavior of PDAC cells in mice?Question肺转移肺转移肺组织病理切片肺组织病理切片Result 5：Modulation of PDGFRb Expression Levels Mediates the Phenotypic E

23、ffects of Mutant p53 Depletion In Vivo转移灶大小转移灶大小免疫组化免疫组化whether pharmacologic inhibition of the PDGFRb pathway recapitulates the effects of PDGFRb or mutant p53 depletion?时效关系实验时效关系实验量效关系实验量效关系实验Crenolanib两种细胞两种细胞IC50whether crenolanib can suppress metastasis?Crenolanib处理与处理与DMSO处理处理荧光和免疫组化荧光和免疫组化细胞

24、凋亡细胞凋亡KPflC 细胞细胞pancreatic cancer cells can provide a source of PDGF ligand that could trigger autocrine activation of PDGFRb条件培养条件培养Summary 5abrogation of autocrine activation signaling of PDGFRb leads to a significant reduction of invasion and metastasis driven by mutant p53.whether PDGFRb inhibit

25、ion prevents the development of metastasis in KPC mice?Question量效关量效关系系抑制细胞增殖所需抑制细胞增殖所需IC50体外体外KPC 细胞侵袭实验细胞侵袭实验Imatinib处理处理KPC的肺转移的肺转移Result 5：Imatinib Inhibits the Development of Metastases in a PDAC Mouse Model by Targeting PDGFRbthe high disease burden in the pancreas was the primary cause of dea

26、th肿瘤体积肿瘤体积总体存活率总体存活率92%15%其他器官转移情况其他器官转移情况不同器官不同器官HE染色染色DAPI,blue;CK8,red;pPDGFRb,green imatinib was able to effectively inhibit PDGFRb activity in primary tumors based on reduced levels of phospho-PDGFRb in the tumor cellspPDGFRb强度强度imatinib对体内对体内PDGFRb的影响的影响Summary 6by inhibiting the kinase activi

27、ty of PDGFRb,imatinib significantly diminishes the metastatic potential of pancreatic cancer cells.whether upregulation of PDGFRb is correlated with prognosis or with the clinicopathological characteristics of PDACs in patients.QuestionResult 5：Imatinib Inhibits the Development of Metastases in a PD

28、AC Mouse Model by Targeting PDGFRb不同种类肿瘤中Mutp53敲除KPflC cell:a p53 null cell line不同种类肿瘤中Mutp53敲除突变体能够通过内源性的促进胰腺癌的转移Mutant p53 maintain metastatic phenotypePDGFRb Luciferase Reporter AssayPDGFRb as a Downstream Mediator of Mutantp53不同种类肿瘤中Mutp53敲除whether pharmacologic inhibition of the PDGFRb pathway

29、recapitulates the effects of PDGFRb or mutant p53 depletion?angiogenesiswhether PDGFRb levels correlate with the status of p53?（优选）突变体能够通过内源性的促进胰腺癌的转移the high disease burden in the pancreas was the primary cause of deathResult 4：Mutant p53 Disrupts the p73/NF-Y Complex to Mediate PDGFRb Expression a

30、nd Tumor Cell InvasionMouse StudiesWound Healing and Invasion Assayswhether upregulation of PDGFRb is correlated with prognosis or with the clinicopathological characteristics of PDACs in patients.Coimmunoprecipitation and Chromatin ImmunoprecipitationWound Healing and Invasion Assays无病存活率无病存活率不同阶段不

31、同阶段PDGFRb表达表达血管内渗血管内渗whether PDGFRb levels correlate with the status of p53?Result 7：PDGFRb Expression Correlates with Disease-free Survival in Human Pancreatic,Colorectal,and Ovarian Cancer Patients磷酸化磷酸化PDGFRb强度强度p53积累积累结直肠癌无病生存率结直肠癌无病生存率As in PDAC,a significant increase in PDGFRb expression was o

32、bserved in higher-stage colorectal不同分期不同分期PDGFRb的表达水平的表达水平结直肠癌结直肠癌卵巢癌卵巢癌p53-/-细胞过表达PDGFRbthe high disease burden in the pancreas was the primary cause of deathRNA Sequencing and Data AnalysisMutant p53 maintain metastatic phenotypeResult 5：Modulation of PDGFRb Expression Levels Mediates the Phenotyp

33、ic Effects of Mutant p53 Depletion In Vivo抑制细胞增殖所需IC50PDGFRb madiates metastasis when Mutant p53 was depleted（优选）突变体能够通过内源性的促进胰腺癌的转移Result 5：Modulation of PDGFRb Expression Levels Mediates the Phenotypic Effects of Mutant p53 Depletion In Vivomutant p53 promotes invasion,inpart,depends on its abilit

34、y to enhance PDGFRb expression through the disruption of the inhibitory p73/NF-Y complexthe high disease burden in the pancreas was the primary cause of deaththe high disease burden in the pancreas was the primary cause of death抑制细胞增殖所需IC50p73 can repress the transcription of PDGFRbResult 5：Imatinib

35、 Inhibits the Development of Metastases in a PDAC Mouse Model by Targeting PDGFRb不同种类肿瘤中Mutp53敲除不同种类肿瘤中Mutp53敲除75%p53 mutantmutant p53 can affect p73/NF-YBPDGFRb Luciferase Reporter Assay磷酸化PDGFRb强度PDGFRb as a Downstream Mediator of Mutantp53Wound Healing and Invasion AssaysCrenolanib处理与DMSO处理KPflC

36、cell:a p53 null cell lineangiogenesisPDGFRb Luciferase Reporter Assaywhether the repressive action of p73 on PDGFRb transcription was mediated by NF-Y and modulated by mutant p53?pancreatic cancer cells can provide a source of PDGF ligand that could trigger autocrine activation of PDGFRbby inhibitin

37、g the kinase activity of PDGFRb,imatinib significantly diminishes the metastatic potential of pancreatic cancer cells.mutant p53 can affect p73/NF-YBmutp53/PDGFRb 分别敲除，A2.RNA Sequencing and Data Analysis不同种类肿瘤中Mutp53敲除the high disease burden in the pancreas was the primary cause of deathThe invasive

38、ness depend on mutant p53.mutant p53 can affect p73/NF-YBthe high disease burden in the pancreas was the primary cause of death（优选）突变体能够通过内源性的促进胰腺癌的转移the high disease burden in the pancreas was the primary cause of deathSummary 7elevated PDGFRb expression correlated significantly with the status of p53,higher tumor stage,and a poorer disease-free survival rate in pancreatic,colorectal,and ovarian cancer patients.