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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  • Background image from 03/20/2007, IL-6 in green
  • Talking points:Senescence is now a well-established in vivo tumor suppressor that rivals apoptosis in significance
  • Talking points:
  • Talking points: -ATM/ATR knockdown prevents the SASP-transient DNA damage response does not activate the SASP, only “persistent” response
  • Talking points:-one of the most effective and reproducible ways to induce genotoxic stress is to damage a cell’s DNA with ionizing radiation. We call this XRA-induced senescence, or more generally, DNA-damage-induced senescence.-the cells stop growing immediately and, over the next 10 days, become fully senescent, SASP included.-looked at p38-P levels (and one of its downstream targets, Hsp27) and found that they increase at roughly the same rate that IL-6 levels do. This was promising.Data:-growth curve from 09/23/2008 – 0.05x shRelA Rd3-IL-6 ELISA from 10/22/2007-timecourse western from 11/20/2007
  • Talking points:-p38 inhibitor SB203580 inhibits downstream p38 signaling without affecting p38-P levels. Does this by outcompeting ATP for the p38MAPK binding pocket. Hsp27 is a known downstream target of p38 and is widely used as a downstream marker of p38MAPK activity. It plays no known role in senescence.-through testing, I determined that 48 hours was sufficient to achieve maximal p38 inhibition and maximal effect on the SASP.-we examined IL6, IL8, and GMCSF because they are, in general, 3 of the most consistently upregulated SASP factors. Data:- +/- SB western from 09/18/2008 IL6 IF from 02/20/2008, Wi38s (contrast adjusted image from p38 paper folder) composite ELISA from Stat Analysis for paper.xlsx (actual data from IL6:08/27/2007, IL8:10/22/2007, GM-CSF:10/1/2007)
  • Talking points:-cytokine array shows marked down-regulation with 48 hours of p38 inhibition in already-senescent cells-hierarchical clustering-9/10 of top 10 factors affected by p38 inhibition-p38MAPK inhibition can mitigatean important biological consequence of the SASP, i.e. stimulation of epithelial cell invasion-had to use p38shRNAs because a chemical inhibitor in the media would have had undesirable effects on the cancer cellsData:-antibody array data from 08/06/2009-p38alpha IL6 ELISA data from 12/04/2008-invasion assay data from 02/06/2009
  • Talking points:-oncogene expression is another common method for inducing senescence, and the most common oncogene is a constitutively active form of Ras, RasV12. when overexpressed, it causes a burst of hyperproliferation that leads to DNA damage and ROS generation, activating the senescence response. Despite being more indirect than DDIS, many argue it is more physiologically relevant.-we first determined that, as in DDIS, Ras-induced senescence activates p38MAPK. p16 is a marker of senescence – it is a key player in the growth arrest pathway. For our purposes, if p16 is high, a cell is senescent.-antibody array shows marked down-regulation with 48 hours of p38 inhibition in already-senescent cells-hierarchical clustering-9/10 of top 10 factors affected by p38 inhibitionData:-RAS western from 03/18/2009 – RAS+shRelA Rd1-RAS growth curve from 02/16/2009 – RAS+SB Rd1-RAS array data from 06/09/2009 – RAS+SB Rd2
  • Talking points:-MKK6 is the upstreamkinase of p38MAPK. So far as we know, it only phosphorylates p38. MKK6EE is a constitutively active form. When you express MKK6EE in fibroblasts, you get constitutive activation of endogenous p38, seen in the western. this cannot be prevented by the p38 inhibitor for the reason discussed earlier, but the inhibitor does prevent the downstream effects: as you may remember from my introduction to p38, constitutive p38 activity can induce senescence growth arrest, and p16 is a marker of that. Its increase is markedly diminished by p38 inhibition, to such an extent that there is no decrease in growth rate. All this does it make us reasonably confident that the effects of MKK6 are operating through p38.-MKK6EE expression induces a SASPData:MKK6 Western from 12/19/2008MKK6 growth curve from 12/19/2008MKK6 array data from 06/09/2009
  • Talking points:-of the top 10 SASP factors in XRA and Ras senescence, most (70% and 90%, respectively), are induced by constitutive p38 activity-does this without inducing genotoxic stress – there’s no hyperproliferation or DNA damage associated with MKK6 expression. If you look at 53BP1 foci, which are generally present in senescent cells and are markers of persistent genotoxic stress, you see almost no increase in MKK6EE cellsData:-MKK6 SASP tables from 06/09/2009-53BP1 composite graph from 12/19/2008-53BP1 example images from 03/20/2007, saved as individual jpgs
  • Talking points:-of course, it was also possible that p38 inhibition was somehow inhibiting the DNA damage response – preventing the cell from sensing genotoxic stress and diminishing the SASP that way. However, several lines of evidence point against that. 1) there is no affect on the phosphorylation status of DDR proteins ATM, Chk2, p53-Ser15, etc that are required for the SASP, 2) there is no affect on the formation or resolution of 53BP1 foci, 3) there is no affect on the growth arrest, which also depends on sensing of genotoxic stress, after irradiation (data not shown)Data:-DNA damage timecourse western from 11/23/2008-53BP1 timecourse graph from 9/12/2008
  • Talking points:-So how does p38 inhibition work to decrease SASP protein levels?-could theoretically affect any part of the route to eventual secretion, including transcription, stability, translation, degradation, secretion, etc-we look at mRNA levels of 5 of the most upregulated, p38 sensitive SASP genes – they were down, similar to protein levels-for a more quantitative analysis, we compared the fold decrease of protein and mRNA levels in SEN(XRA) cells with p38 inhibition. The fold decreases matched. this of course doesn’t rule out p38MAPK having an effect on post-mRNA processes like translation, degradation, secretion, etc, but it suggests that most of the effect of p38MAPK inhibition comes from changing mRNA abundance. This could be due to changes in transcription, or changes in mRNA stability.Data:-TaqMan heat map from 05/21/2008-mRNA vs ELISA graph from 05/21/2008, ELISA data the same as IL6,IL8,GMCSF ELISA graph in Fig 1
  • Talking points:-performed a bioinformatics search for overrepresented TFBS in the promoters of p38-regulated SASP genes. To make this a more stringent search, we only used those genes that were upregulated by constitutive p38 activation. -queried all the TF weight matrices in the TRANSFAC database and sorted by statistical significance of overrepresentation. Looked between -200 and 0-NF-kB came out on top, follow closely by C/EBP, another transcription factor that often associates with the NF-kB transcriptional complex.-The TF’s repeat in this list because the same transcription factor can correspond to more than one weight matrix in the database.Data:-TFBS table from Data Resources --> SASP genes sequences -->TFM Explorer Analysis --> TFM Analysis - MKK6EE cumulative.xlsx
  • Picture from Wikipedia (NF-kB article)Talking points:-there are 3 main ways of regulating the activity of the NF-kB transcriptional complex:Nuclear translocationRelA DNA bindingCoactivators allowing for full transcriptional activity
  • Talking points:-RelA nuclear localization increased at senescence, not affected by p38 inhibition-NF-kB DNA binding increased in all types of senescence, not affected by p38 inhibition-timecourse shows a rate of DNA binding increase that correlates well with p38 phosphorylation after irradiation.-this led us to the last step of NF-kB regulation: actual transcriptional output, determined by the full formation of the complex.Data:-RelA IF from 05/22 --> Files for Paper (PRE and SEN(XRA))and from 07/04 --> Files for Paper (SEN(XRA)+SB) -NF-kB DNA binding activity composite graph from 03/22/2009-NF-kB DNA binding activity timecourse graph from Stat Analysis for paper.xlsx, data from 10/22/2008
  • Talking points:-p38 inhibition significantly decreases NF-kB transcriptional activity, as measured by a luciferase reporter construct, suggesting it operates through some coactivator-interestingly, knockdown of the DDR proteins that are required for SASP activity also decreases NF-kB transcriptional activity.-of course, you may be thinking that DDR knockdown simply prevents p38 phosphorylation, but it turns out that this isn’t the case. Knockdown of ATM has no effect on p38-P levels, leading us to conclude that, while these two pathways both act on NF-kB, they do so through independent pathways.p38MAPk inhibition seems to only affect the output of an NF-kB reporter (not nuclear translocation or DNA binding), suggesting it operates through some coactivator of the complexData:-SEN(XRA) +/- SB NF-kB reporter graph from 06/18/2009-SEN(XRA) +/-shATM, shNBS NF-kB reporter graph from 07/14/2009-shATM western from 11/26/2008
  • Talking points:-shRelA knocks down RelA, the most common and constitutively expressed family member, without affecting RelB or C-Rel.-knocks down most of the SASP in SEN(XRA), and the few SASP markers I’ve tested in SEN(RAS)Data:-shRelA western from 08/22/2008, PRE Wi38-HCA2 SEN(XRA)shRelA Array data from 12/18/2008, actual figure from 08/06/2009-HCA2 SEN(RAS) shRelA composite ELISA graph from 05/05/2009
  • Talking points:-significant overlap; of the top 10 factors that require p38MAPK, 100% are RelA dependentData:-HCA2 SEN(XRA)shRelA Array data from 12/18/2008, actual figure from 08/06/2009
  • Talking points:-both p38MAPK pathway and NF-kB pathway are meant to be rapid-response pathways. The fact that they are not activated immediately after a senescence-inducing stimulus suggests there is an active repression going on in the cell. Evolutionary theory: gives the cell time to repair its DNA before signaling to its surroundings that it is damaged.
  • 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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