Zhiwei Wang, Sanjeev Banerjee, Dejuan Kong, Yiwei Li and Fazlul H. Sarkar Department of Pathology, Karmanos Cancer Institu...
Introduction
Pancreatic Cancer (PC) <ul><li>High mortality, the fourth leading cause of cancer-related death in the USA </li></ul><ul><...
Forkhead box M1 (FOXM1) <ul><li>A transcription factor </li></ul><ul><li>Involved in cell proliferation and apoptosis </li...
Transcription factor <ul><li>Controls transcription </li></ul><ul><li>DNA binding domains (DBDs) </li></ul><ul><li>Promoti...
www.cs.uiuc.edu
Forkhead Box (Fox) <ul><li>Superclass: Helix-turn-helix </li></ul><ul><li>Class: Fork head </li></ul>wikipedia.org http://...
FOXM1 regulate transcription of many cell cycle genes (Cdc25A, Cdc25B, cyclin B, cyclin D1, p21, p27) http://mcb.asm.org
Angiogenesis ( 血管新生 ) Cell and molecular biology, Gerald Karp, p.685 http://ir.progenics.com
Vascular endothelial growth factor (VEGF) http://www.researchvegf.com FoxM1 regulates VEGF signaling in various cell types
Migration and Invasion Migration ( 遷移 ) Invasion ( 入侵 )
Cell and molecular biology, Gerald Karp, p.240, p.244 Type IV collagen network
Matrix metalloproteinase(MMP) <ul><li>Endopeptidases </li></ul><ul><li>Capable of degrading all kinds of extracellular mat...
Previous research <ul><li>Alterations in FoxM1 signaling are associated with tumorigenesis </li></ul><ul><li>Overexpressio...
The aim in this article Whether FoxM1 could be an effective therapeutic target for PC ?
Methods and Results <ul><li>To test the consequences of down-regulation and up-regulation of FoxM1 in PC, using: </li></ul...
Experimental Design MTT test Flow cytometry ELISA Angiogenesis assays Migration and Invasion assay
RNA interference (RNAi) wikipedia.org
Gene transfection FoxM1 siRNA, siRNA control or FoxM1 cDNA plasmid Lipofectamine 2000
PC Cells Incubate the cells
PC Cell DNA
To determine the baseline expression of FoxM1 in PC cell lines Fig.1
To examine the relative mRNA levels of FoxM1 in three PC cell lines Fig.1
http://biochemistry.yonsei.ac.kr/
Real time 基因工程技術原理 ( 實驗講義 ), 12-12 <ul><li>TaqMan probe:  是一段 oligonucleotide ,可接合位於 forward 和 reverse primer 間之序列 </li></...
To determine the efficacy of FoxM1 siRNA for knockdown FoxM1 mRNA and protein Fig.1 CS : control siRNA FS : FoxM1 siRNA
To examine the effect of FoxM1 siRNA on cell growth of PC cells Fig.2 wikipedia.org MTT test
To examine the effect of FoxM1 cDNA on cell growth of PC cells CP : control plasmid FP : FoxM1 cDNA plasmid Fig.1D Fig.2
<ul><li>Down-regulation of FoxM1 expression by siRNA inhibited cell growth </li></ul><ul><li>Overexpression of FoxM1 by cD...
To further investigate the growth-inhibitory effect of FoxM1 knockdown in PC cells Fig.3 Cell cycle anatysis
To examine the level of expression of several known G 0 -G 1  cell cycle regulatory factors Fig.4
<ul><li>Down-regulation of FoxM1 decreased cell population in the S phase </li></ul>
To investigate whether MMP-9 and MMP-2 were down-regulated by FoxM1 siRNA in PC cell lines Fig.5
To examine whether the down-regulation of FoxM1 could lead to a decrease in MMP-2 and MMP-9 activity in PC cells Fig.5 ELISA
<ul><li>Down-regulation of FoxM1 decreased MMP-2 and MMP-9 gene transcription and their activities </li></ul><ul><li>Overe...
To investigate whether FoxM1 siRNA has any effect on reducing the level of uPAR (urokinase-type plasminogen activator rece...
To test the effects of FoxM1 down-regulation on PC cell migration and invasion, using BD BioCoat Tumor Invasion Assay Syst...
Fig.6 Cell migration and invasion assay
Stain http://www.dojindo.cn wikipedia.org Calcein-AM Calcein 細胞內酯酶水解
<ul><li>Down-regulation of FoxM1 decreased PC cell migration and invasion </li></ul><ul><li>Up-regulation of FoxM1 increas...
To explore whether FoxM1 siRNA reduced VEGF activity Fig.5 FoxM1 siRNA decreased VEGF acivity (Overexpression FoxM1 increa...
To test whether conditioned media from FoxM1 siRNA-transfected cells could reduce the tube formation Fig.6 Matrigel in vit...
<ul><li>Reduced tube formation of HUVECs induced by conditioned media from FoxM1 siRNA-transfected cells </li></ul><ul><li...
Summary
Conclusion The down-regulation of   FoxM1 could potentially be an effective therapeutic approach   for the inactivation MM...
Discussion <ul><li>FoxM1 gene is up-regulated in PC due to transcriptional regulation by the Sonic Hedgehog ( 音猬因子 ) pathw...
wikipedia.org
 
Urokinase-type plasminogen activator (uPA) <ul><li>Important genes in the processes of tumor cell invasion and metastasis ...
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Down regulationof forkheadboxm1transcriptionfactorleadstotheinhibitionofinvasionandangiogenesisofpancreaticcancercellsppt

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Down-regulation of Forkhead Box M1 Transcription Factor Leads to the Inhibition of Invasion and Angiogenesis of Pancreatic Cancer Cells.

Zhiwei Wang, Sanjeev Banerjee, Dejuan Kong, Yiwei Li, and Fazlul H. Sarkar

Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan

Cancer Res 2007; 67: (17). September 1, 2007

Published in: Health & Medicine
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Transcript of "Down regulationof forkheadboxm1transcriptionfactorleadstotheinhibitionofinvasionandangiogenesisofpancreaticcancercellsppt"

  1. 1. Zhiwei Wang, Sanjeev Banerjee, Dejuan Kong, Yiwei Li and Fazlul H. Sarkar Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan Design by ChauChanLao 2009.03 Cancer Res 2007; 67: (17). September 1, 2007 IF: 7.672
  2. 2. Introduction
  3. 3. Pancreatic Cancer (PC) <ul><li>High mortality, the fourth leading cause of cancer-related death in the USA </li></ul><ul><li>No effective methods for early diagnosis </li></ul><ul><li>Lack effective therapies </li></ul><ul><li>Symptoms : pain in the upper abdomen, loss of appetite, significant weight loss, jaundice </li></ul>wikipedia.org
  4. 4. Forkhead box M1 (FOXM1) <ul><li>A transcription factor </li></ul><ul><li>Involved in cell proliferation and apoptosis </li></ul><ul><li>Affects the development and function of many organs </li></ul><ul><li>A key cell cycle regulator of both the transition from G 1 to S phase and the progression to mitosis </li></ul>
  5. 5. Transcription factor <ul><li>Controls transcription </li></ul><ul><li>DNA binding domains (DBDs) </li></ul><ul><li>Promoting or blocking the recruitment of RNA polymerase to specific genes </li></ul>
  6. 6. www.cs.uiuc.edu
  7. 7. Forkhead Box (Fox) <ul><li>Superclass: Helix-turn-helix </li></ul><ul><li>Class: Fork head </li></ul>wikipedia.org http://departments.oxy.edu/biology/biology/
  8. 8. FOXM1 regulate transcription of many cell cycle genes (Cdc25A, Cdc25B, cyclin B, cyclin D1, p21, p27) http://mcb.asm.org
  9. 9. Angiogenesis ( 血管新生 ) Cell and molecular biology, Gerald Karp, p.685 http://ir.progenics.com
  10. 10. Vascular endothelial growth factor (VEGF) http://www.researchvegf.com FoxM1 regulates VEGF signaling in various cell types
  11. 11. Migration and Invasion Migration ( 遷移 ) Invasion ( 入侵 )
  12. 12. Cell and molecular biology, Gerald Karp, p.240, p.244 Type IV collagen network
  13. 13. Matrix metalloproteinase(MMP) <ul><li>Endopeptidases </li></ul><ul><li>Capable of degrading all kinds of extracellular matrix proteins </li></ul>wikipedia.org The main substrates of the gelatinases are type IV collagen and gelatin It has reported that MMP-9 expression is elevated in the liver of FoxM1B trangenic mice
  14. 14. Previous research <ul><li>Alterations in FoxM1 signaling are associated with tumorigenesis </li></ul><ul><li>Overexpression of FOXM1 has been found in a variety of aggressive human carcinomas including pancreatic cancer </li></ul><ul><li>FoxM1 signaling is known to control cell migration and invasion, which are important processes involved in tumor development and metastasis </li></ul><ul><li>The precise role and the molecular mechanism of action of FoxM1 in PC remain unclear </li></ul>
  15. 15. The aim in this article Whether FoxM1 could be an effective therapeutic target for PC ?
  16. 16. Methods and Results <ul><li>To test the consequences of down-regulation and up-regulation of FoxM1 in PC, using: </li></ul><ul><li>siRNA </li></ul><ul><li>cDNA </li></ul>
  17. 17. Experimental Design MTT test Flow cytometry ELISA Angiogenesis assays Migration and Invasion assay
  18. 18. RNA interference (RNAi) wikipedia.org
  19. 19. Gene transfection FoxM1 siRNA, siRNA control or FoxM1 cDNA plasmid Lipofectamine 2000
  20. 20. PC Cells Incubate the cells
  21. 21. PC Cell DNA
  22. 22. To determine the baseline expression of FoxM1 in PC cell lines Fig.1
  23. 23. To examine the relative mRNA levels of FoxM1 in three PC cell lines Fig.1
  24. 24. http://biochemistry.yonsei.ac.kr/
  25. 25. Real time 基因工程技術原理 ( 實驗講義 ), 12-12 <ul><li>TaqMan probe: 是一段 oligonucleotide ,可接合位於 forward 和 reverse primer 間之序列 </li></ul><ul><li>R: reporter fluorochrome </li></ul><ul><li>Q: quencher fluorochrome </li></ul>
  26. 26. To determine the efficacy of FoxM1 siRNA for knockdown FoxM1 mRNA and protein Fig.1 CS : control siRNA FS : FoxM1 siRNA
  27. 27. To examine the effect of FoxM1 siRNA on cell growth of PC cells Fig.2 wikipedia.org MTT test
  28. 28. To examine the effect of FoxM1 cDNA on cell growth of PC cells CP : control plasmid FP : FoxM1 cDNA plasmid Fig.1D Fig.2
  29. 29. <ul><li>Down-regulation of FoxM1 expression by siRNA inhibited cell growth </li></ul><ul><li>Overexpression of FoxM1 by cDNA transfection promoted cell growth </li></ul>
  30. 30. To further investigate the growth-inhibitory effect of FoxM1 knockdown in PC cells Fig.3 Cell cycle anatysis
  31. 31. To examine the level of expression of several known G 0 -G 1 cell cycle regulatory factors Fig.4
  32. 32. <ul><li>Down-regulation of FoxM1 decreased cell population in the S phase </li></ul>
  33. 33. To investigate whether MMP-9 and MMP-2 were down-regulated by FoxM1 siRNA in PC cell lines Fig.5
  34. 34. To examine whether the down-regulation of FoxM1 could lead to a decrease in MMP-2 and MMP-9 activity in PC cells Fig.5 ELISA
  35. 35. <ul><li>Down-regulation of FoxM1 decreased MMP-2 and MMP-9 gene transcription and their activities </li></ul><ul><li>Overexpression of FoxM1 by cDNA transfection led to an increase in MMP-2 and MMP-9 expression and activity (data not shown) </li></ul>
  36. 36. To investigate whether FoxM1 siRNA has any effect on reducing the level of uPAR (urokinase-type plasminogen activator receptor), which are able to regulate the MMP-9 activity in PC Fig.5 FoxM1 siRNA reduced uPAR gene transcription and translation
  37. 37. To test the effects of FoxM1 down-regulation on PC cell migration and invasion, using BD BioCoat Tumor Invasion Assay System www.bdbiosciences.com
  38. 38. Fig.6 Cell migration and invasion assay
  39. 39. Stain http://www.dojindo.cn wikipedia.org Calcein-AM Calcein 細胞內酯酶水解
  40. 40. <ul><li>Down-regulation of FoxM1 decreased PC cell migration and invasion </li></ul><ul><li>Up-regulation of FoxM1 increased PC cell migration and invasion (data not shown) </li></ul>
  41. 41. To explore whether FoxM1 siRNA reduced VEGF activity Fig.5 FoxM1 siRNA decreased VEGF acivity (Overexpression FoxM1 increased VEGF acivity, data not shown)
  42. 42. To test whether conditioned media from FoxM1 siRNA-transfected cells could reduce the tube formation Fig.6 Matrigel in vitro HUVECs tube formation assay
  43. 43. <ul><li>Reduced tube formation of HUVECs induced by conditioned media from FoxM1 siRNA-transfected cells </li></ul><ul><li>Increased tube formation of HUVECs induced by conditioned media from FoxM1 cDNA-transfected cells (data not shown) </li></ul>
  44. 44. Summary
  45. 45. Conclusion The down-regulation of FoxM1 could potentially be an effective therapeutic approach for the inactivation MMP-2, MMP-9, and VEGF, which is likely to result in the inhibition of cell growth, migration, invasion, angiogenesis, and metastasis of PC.
  46. 46. Discussion <ul><li>FoxM1 gene is up-regulated in PC due to transcriptional regulation by the Sonic Hedgehog ( 音猬因子 ) pathway </li></ul><ul><li>Sonic hedgehog homolog (SHH) is one of three proteins in the mammalian hedgehog ( 刺猬因子 ) family </li></ul><ul><li>It plays a key role in regulating vertebrate organogenesis </li></ul><ul><li>SHH remains important in the adult. It controls cell division of adult stem cells </li></ul><ul><li>SHH is involved in vascularization of certain embryonic tissues as well as in inducing angiogenesis in an adult mammalian system </li></ul>www.bdbiosciences.com
  47. 47. wikipedia.org
  48. 49. Urokinase-type plasminogen activator (uPA) <ul><li>Important genes in the processes of tumor cell invasion and metastasis </li></ul><ul><li>Play important roles in many aspects of the angiogenic process, including extracellular cascade of proteolysis, basement membrane degradation, cell migration, and invasion </li></ul><ul><li>Cancer cells with higher levels of uPA or uPAR will tend to invade surrounding tissues andsubsequently migrate to blood vessels. </li></ul>

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