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Tumor suppression and inflammation:<br />Controlling the senescence secretory phenotype<br />Adam Freund1,2,3 , Christophe...
Tumor Suppressor mechanisms falls into two general classes<br />
Cellular senescence is caused by potentially oncogenic stimuli<br />Cellular senescence: irreversible cell cycle arrest<br...
Senescence occurs in vivo in response to oncogenic mutations<br />Senescence markers<br />Michaloglou, C, et al. (2005). B...
The two sides of senescence<br />Irreversible<br />Growth <br />Arrest<br />Inflammatory<br />Secretory<br />Profile<br />...
The Senescence-Associated Secretory Phenotype (SASP) <br />is activated by genotoxic stress<br />DNA damage<br />Hyper-rep...
p38MAPK activation is associated with DNA damage-induced senescence<br />Ionizing radiation (XRA)<br />Growth Curve<br />P...
p38MAPK activity is required for markers of the SASP<br />IL-6<br />DAPI<br />PRE<br />PRE<br />+p38 inhibitor<br />SEN(XR...
p38MAPK activity is required for expression of most SASP factors<br />SB = SB203580 = p38 inhibitor<br />
p38MAPK activity is also required for the Ras-induced SASP<br />Constitutive expression of RasV12<br />Empty<br />Vector<b...
p38MAPK activity is sufficient to induce a SASP<br />MKK6<br />Empty Vector<br />MKK6EE<br />MKK6<br />p38-P<br />p38<br /...
p38MAPK activity induces a SASP and is downstream of genotoxic stress<br />53BP1 foci<br />53BP1 foci<br />PRE<br />SEN<br />
p38MAPK inhibition does not affect the DNA damage response<br />PRE<br />Hours after irradiation:<br />PRE<br />2    4   8...
p38MAPK inhibition modulates the SASP at the mRNA level<br />Quantitative RT-PCR<br />Protein versus mRNA level<br />2 mai...
NF-κB binding sites are overrepresented in the promoters of p38MAPK-induced genes<br />
The NF-κB Pathway<br />
NF-κB is activated at senescence<br />RelA<br />DAPI<br />PRE<br />SEN(XRA)<br />PRE<br />SEN(XRA)<br />+p38 inhibitor<br ...
NF-κB transcriptional activity is p38MAPK-dependent<br />SEN(XRA)<br />PRE<br />Genotoxic stress<br />-<br />-<br />+<br /...
RelA is required for the majority of the SASP<br />shRelA #1<br />shRelA #2<br />shGFP<br />RelA<br />RelB<br />C-Rel<br /...
The p38MAPK and RelA-dependent SASP factors overlap<br />The top 10 SASP factors, and their dependence on RelA, p38MAPK, a...
Model:  The SASP pathways<br />Senescence-inducing stimulus <br />(DNA damage, oncogene expression)<br />p38<br />ATM<br /...
Lawrence Berkeley National Laboratory<br />Acknowledgments<br />Judy Campisi<br />Present lab members<br />Pierre Desprez<...
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Tumor suppression and inflammation: controlling the senescence associated secretory phenotype

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This is the powerpoint presentation from a talk I gave at a conference in October, 2009. It will be hard to follow without the spoken part, but it will hopefully give anyone who is interested a brief introduction to my thesis research.

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Tumor suppression and inflammation: controlling the senescence associated secretory phenotype

  1. 1. Tumor suppression and inflammation:<br />Controlling the senescence secretory phenotype<br />Adam Freund1,2,3 , Christopher K. Patil2,3, Shruti Waghray1, Judith Campisi2,3<br />1. Department of Molecular and Cell Biology, University of California, Berkeley, CA<br />2. Buck Institute for Age Research, Novato CA<br />3. Lawrence Berkeley National Laboratory, Berkeley CA<br />
  2. 2. Tumor Suppressor mechanisms falls into two general classes<br />
  3. 3. Cellular senescence is caused by potentially oncogenic stimuli<br />Cellular senescence: irreversible cell cycle arrest<br />Irreversible<br />cell cycle<br /> arrest<br />
  4. 4. Senescence occurs in vivo in response to oncogenic mutations<br />Senescence markers<br />Michaloglou, C, et al. (2005). BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720-724<br />
  5. 5. The two sides of senescence<br />Irreversible<br />Growth <br />Arrest<br />Inflammatory<br />Secretory<br />Profile<br />-Cell autonomous tumor suppression<br />-Alteration of the microenvironment<br />-Stimulation of malignant phenotypes<br />-Global tissue degeneration<br />
  6. 6. The Senescence-Associated Secretory Phenotype (SASP) <br />is activated by genotoxic stress<br />DNA damage<br />Hyper-replication<br />ROS<br />DNA damage response<br />But this is an extremely <br />rapid response (&lt;1 hour)<br />?<br />ATM/NBS1/Chk2<br />SASP<br />And this takes days to develop<br />What other molecular events need to happen?<br />
  7. 7. p38MAPK activation is associated with DNA damage-induced senescence<br />Ionizing radiation (XRA)<br />Growth Curve<br />PRE<br />8-10 days<br />SEN(XRA)<br />Days after irradiation: PRE 2 4 6 8 10<br />p38-P<br />p38<br />Hsp27-P<br />p38MAPK is a mitogen activated protein kinase and part of a well-known stress-response pathway<br />Hsp27<br />Tubulin<br />
  8. 8. p38MAPK activity is required for markers of the SASP<br />IL-6<br />DAPI<br />PRE<br />PRE<br />+p38 inhibitor<br />SEN(XRA)<br />SEN(XRA)<br />+p38 inhibitor<br />
  9. 9. p38MAPK activity is required for expression of most SASP factors<br />SB = SB203580 = p38 inhibitor<br />
  10. 10. p38MAPK activity is also required for the Ras-induced SASP<br />Constitutive expression of RasV12<br />Empty<br />Vector<br />SEN<br />(RAS)<br />PRE<br />p38-P<br />p38<br />8-10 days<br />Hsp27-P<br />Hsp27<br />p16<br />Ras<br />SEN(RAS)<br />Tubulin<br />SB = SB203580 = p38 inhibitor<br />
  11. 11. p38MAPK activity is sufficient to induce a SASP<br />MKK6<br />Empty Vector<br />MKK6EE<br />MKK6<br />p38-P<br />p38<br />p38<br />p16<br />Tubulin<br />MKK6EE = constitutively active form of MKK6 = constitutively active endogenous p38<br />SB = SB203580 = p38 inhibitor<br />
  12. 12. p38MAPK activity induces a SASP and is downstream of genotoxic stress<br />53BP1 foci<br />53BP1 foci<br />PRE<br />SEN<br />
  13. 13. p38MAPK inhibition does not affect the DNA damage response<br />PRE<br />Hours after irradiation:<br />PRE<br />2 4 8 24 48 72<br />2 4 8 24 48 72<br />p38 inhibitor: - - - - - - - + + + + + + +<br />ATM-P<br />ATM<br />Chk2-P<br />Chk2<br />p53-P (Ser15)<br />p53 total<br />p21<br />Tubulin<br />
  14. 14. p38MAPK inhibition modulates the SASP at the mRNA level<br />Quantitative RT-PCR<br />Protein versus mRNA level<br />2 main ways to modulate mRNA levels:<br />Stability<br />Transcription<br />SB = SB203580 = p38 inhibitor<br />
  15. 15. NF-κB binding sites are overrepresented in the promoters of p38MAPK-induced genes<br />
  16. 16. The NF-κB Pathway<br />
  17. 17. NF-κB is activated at senescence<br />RelA<br />DAPI<br />PRE<br />SEN(XRA)<br />PRE<br />SEN(XRA)<br />+p38 inhibitor<br />RelA = most common NF-kB subunit<br />Time after XRA:<br />
  18. 18. NF-κB transcriptional activity is p38MAPK-dependent<br />SEN(XRA)<br />PRE<br />Genotoxic stress<br />-<br />-<br />+<br />+<br />shATM:<br />ATM<br />p38<br />ATM<br />p38-P<br />p38<br />Tubulin<br />NF-κB<br />
  19. 19. RelA is required for the majority of the SASP<br />shRelA #1<br />shRelA #2<br />shGFP<br />RelA<br />RelB<br />C-Rel<br />Tubulin<br />o3<br />
  20. 20. The p38MAPK and RelA-dependent SASP factors overlap<br />The top 10 SASP factors, and their dependence on RelA, p38MAPK, and ATM<br />RelA knockdown<br />SEN(XRA)<br /> SASP<br />p38 inhibition<br />
  21. 21. Model: The SASP pathways<br />Senescence-inducing stimulus <br />(DNA damage, oncogene expression)<br />p38<br />ATM<br />NF-κB<br />Transcription of SASP factors<br />
  22. 22. Lawrence Berkeley National Laboratory<br />Acknowledgments<br />Judy Campisi<br />Present lab members<br />Pierre Desprez<br />Albert Davalos<br />Art Orjalo<br />Chris Patil<br />Remi-Martin Laberge<br />Chris Wiley<br />Bridget Gengler<br />Michael Velarde<br />Ying Zou<br />Past lab members<br />Francis Rodier <br />Jean PhilipppeCoppe<br />Buck Institute for Age Research<br />

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