Insulin/IGF1-IRS-PI-3 kinase-mtor signaling pathway

Transcription

1 Insulin/IGF1-IRS-PI-3 kinase-mtor signaling pathway Nutrients RAPAMYCIN (FKBP12) mtor/rictor complex phosphorylates Akt at the S473 activating site in a Rheb-independent fashion

2 TSC1 or TSC2-null MEFs show reduced IRS1 and decreased insulin activation of Akt that is reversed by rapamycin Rapamycin treatment for 24 h prior to insulin treatment for 5 min This suggests that there is a negative feedback loop requiring mtor

3 Feedback loop reduces insulin signaling in TSC-null cells S6K feedback downstream of mtor leads to reduction IRS1/2 levels in TSC-null cells (also in LKB1-null cells) IRS1 and IRS2 are less stable in TSC1 and TSC2-null cells - proteasome inhibitor treatment partially restores IRS levels indicating that turnover is through a ubiquitin/proteasome-mediated process IRS1/IRS2 mrna levels are reduced in TSC1 and TSC2-deficient cells and are restored by rapamycin treatment, implying that there is also feedback at the level of transcription TSC-deficient cells are insulin resistant and susceptible to apoptotic stimuli due to defective IGF-1 survival signaling Shah et al. Curr Biol 14:1650 (2004)

4 Model for Rheb/mTOR/S6K negative feedback on IRS function Activated Rheb/mTOR/S6K signaling downregulates IRS function at three levels 1. Inhibition of IRS PTB binding to the activated InsR by direct S6K phosphorylation of Ser (S307) in the PTB domain and other sites (S527), and indirectly other S6Kdependent Ser.Pro sites (S312, S616, S636) contribute to downregulation of IRS signaling (fast) 2. Enhanced degradation of IRS1/2, which is likely phosphorylation dependent (slow) (multiple Ser?) 3. Reduced transcription of IRS1/2 genes by an unknown pathway (slow) (nuclear p85 S6K?) NUCLEUS ENERGY DEPLETION Harrington, Lamb et al. JCB 116:213 (2004) Um, Thomas et al. Nature 431:200 (2004) Shah et al. Curr Biol 14:1650 (2004)

5 TSC2-null cells are sensitized to DNA damage and undergo apoptosis Cells in serum free medium for 6 h with or without growth factor Cells in 0.4% serum overnight and then IGF1 for 4 h followed by camptothecin for 24 h Camptothecin or etoposide-induced apoptosis in TSC1 or TSC2-null cells is largely blocked by rapamycin pretreatment for 24 hours

6 Hypersensitivity of TSC-null cells to DNA damage-induced apoptosis is blocked by rapamycin inhibition of mtor The defect in Akt activation and downstream survival responses could be responsible for the sensitivity of TSC-null cells to DNA-damaging agents

7 + 53 /- -p /+ p5 TS C1 + C1 TS /+ 3+ /+ /- 53 /- -p C2 TS TS C2 + /+ p5 3-/( W T) TSC1/2-deficient MEFs show increased sensitivity to TNFα-induced apoptosis as well as DNA damage Control MMS M M TN S Fα M M S TN Fα TNFα+CHX Cleaved Caspase3-Asp175 Cleaved Caspase3-Asp175 TSC2+/+ TSC2-/- TSC2+/+ untreated TSC2+/+ MMS TSC2-/untreated TSC2-/MMS NF-κB induction by cytokines provides an important survival response

8 NF-κB activation pathways in inflammation and survival NF-κB/IκB families RelA = p65 Bcl-X L, ciaps, GADD45, SOD2 Karin and Greten Nat Rev Immunol 5:749 (2005)

9 Reduced NF-κB activation/nf-κb-dependent gene expression in TSC2 - / - MEFs in response to DNA damage and TNFα TSC2 + / + TSC2 - / - TSC2 + / + TSC1 - / - TSC2 - / - NF-κB EMSA (p65/p50) Oct-1 EMSA TSC2 + / + TSC2 - / - p65 TNFα IL LPS MMS Dox IκBα actin NF-κB target genes

10 Negative feedback reduces insulin signaling in TSC-null cells sirna depletion of TSC1/2 or overexpression of Rheb leads to sustained mtor activation

11 sirna depletion of TSC2 or Rheb overexpression in HEK293 cells attenuates NF-κB-dependent gene expression following DNA damage Control sirna TSC2 sirna TSC2 actin Control transfection Rheb-myc myc actin NF-κB target genes

12 sirna depletion of TSC2 or Rheb overexpression in HEK293 cells also attenuates NF-κB activation and NF-κB dependent gene expression following TNFα treatment sirna depletion of TSC2 in U2OS, MDA-MB-468 and MCF7 human cancer cells causes reduced MMS and TNFα induction of NF-κB target genes

13 Reconstitution of TSC2 restores NF-κB activation and NF-κB dependent resistance to DNA damage/tnfα-induced cell death TSC2 + / + TSC2 - / - TSC2 - / - [R] TSC2 - / - [R] + IκBαM TSC2 IκBα actin TSC2 - / - TSC2 - / - [R] TSC2 - / - TSC2 - / - [R] NF-κB EMSA (p65/p50) MMS TNFα IL Dominant-negative IκBαM reverses TSC2 rescue of DNA damage-induced death

14 Loss of TSC2 reduces IKK and p65 activation following DNA damage TSC2 + / + TSC2 - / - MMS (min) IKKα/β-pSer 180/181 IκBα-pSer 32 actin TSC2 + / + TSC2 - / - MMS (min) p65-pser 536 p65

15 ERK1/2, but not Akt, shows sustained activation following DNA damage and TSC2 is required for ERK1/2 activation TSC2 + / + TSC2 - / - MMS (min) perk1/2-pt 183 /Y 185 perk1/2 TSC2 + / + TSC2 - / - TSC2 - / - [R] MMS perk1/2-pt 183 /Y 185 perk1/2 TSC2 + / + TSC2 - / - Unttd. EGF TPA Unttd. EGF TPA perk1/2-pt 183 /Y 185 perk1/2 TSC2 + / + TSC2 - / - TSC2 - / - [R] MMS Akt-pSer 473 Akt p38-pt 180 /Y 182 p38 EGF and TPA activation of ERK is normal TSC2-null cells

16 Rapamycin induces DNA damage resistance and restores NF-κB and ERK1/2 activation in TSC1/2-deficient MEFs TSC1 - / - TSC2 - / - Rap MMS Rap+MMS Control MMS NF-κB EMSA (p65/p50) MMS+Rap TSC2 - / - TSC2 - / - + Rap. MMS (min) perk1/2-pthr 183 /Tyr 185 perk1/2 TSC2 - / - Rapamycin effect confirms that hyperactive mtor blocks DNA damage-induced NF-κB

17 Rapamycin restores TNFα-induced Akt and NF-κB activation while LY attenuates NF-κB signaling TSC2 + / + TSC2 - / - TNFα Rap Untreated Rap TNFα Rap+TNFα Akt-pSer 473 Akt Untreated TNFα TNFα +LY Untreated Rap+TNFα Rap+TNFα +LY NF-κB EMSA (p65/p50) NF-κB EMSA (p65/p50) TSC2 - / - TSC2 + / + TSC2 - / - Akt inhibition blocks TNFα induction of NF-κB target genes

18 mtor hyperactivation inhibits NF-κB activation Loss of TSC function attenuates IKK and NF-κB activation following DNA damage and TNFα treatment, illustrating a new type of crosstalk ERK1/2 shows sustained activation following DNA damage in wild type MEFs. Lack of TSC2 decreases DNA damage but not EGF-induced ERK1/2 activation Inhibition of MEK blocks IKK and NF-κB activation by DNA damage in WT MEFs and reverses rapamycin-induced DNA damage resistance in TSCnull MEFs. PI-3 kinase inhibition reduces NF-κB signaling induced by MMS and TNFα, and reverses rapamycin-induced DNA damage resistance Akt is not significantly activated by DNA damage in MEFs. Treatment with an Akt inhibitor (A ) does not block NF-κB target gene expression or enhance MMS-induced death, implying that Akt activation is not a major survival pathway in cells with DNA damage, and that Akt activation is not needed for NF-κB activation in response to DNA damage Activation of the mtor pathway counteracts the pro-survival effects of NF-κB activation on gene expression in response to DNA damage and cytokines Ghosh et al. Cancer Cell 10:215 (2006)

19 Crosstalk between the PI-3K/Akt/TSC/mTOR pathway and NF-κB activation pathways ATM? ATM? ATM? TNFα-mediated NF-κB activation requires Akt, whereas DNA damage induction of NF-κB requires ERK - the molecular mechanisms through which mtor signaling blocks IKK activation by either pathway is unknown

20 Implications for use of mtor inhibitors in cancer therapy Many cancer cells have hyperactive mtor (e.g. due to PTEN, LKB1, TSC, PI-3K or Akt mutations, etc.), suggesting that mtor inhibitors (e.g. rapamycin analogues) could be a useful cancer therapy Inhibition of mtor in cancer cells with elevated mtor signaling leads to upregulation of signaling via IGF-1 receptor and potentially other RTKs, thereby promoting enhanced Akt survival signaling. This suggests that it may be useful to combine the use of an mtor inhibitor (e.g. rapamycin) with an inhibitor of IGF-1R, PI-3 kinase or Akt in cancer therapy Inhibition of mtor in cancer cells with elevated mtor signaling may also lead to upregulation of NF-κB activation in response to DNA damage (e.g. by chemotherapeutic agents) or cytokines. This suggests that it may be therapeutically effective to combine the use of an mtor inhibitor (e.g. rapamycin) with an inhibitor of NF-κB signaling (e.g. an IKK inhibitor)

21 The long road to the GLEEVEC TM cancer drug 1845 CML 1960 Chr 22Δ Ph Chr 1973 t(9:22) translocation 1982 Bcr-Abl (22:9) 1984 Bcr-Abl PTK activity 1990 Bcr-Abl causes CML in mice 1996 STI571 inhibits CML cell growth and v-abl tumors 1911 RSV 1927 W mutant mouse 1970 Ab-MuLV 1970 v-src gene 1952 polyoma virus 1975 c-src gene 1977 v-src protein 1977 mt antigen 1986 HZ4-FeSV 1978 v-abl protein 1978 v-src PK 1979 mt associated PTK activity 1986 v-kit PTK 1980 v-abl tyrosine kinase 1979 v-src tyrosine kinase 1983 mt associated c-src 1988 W is c-kit 1988 First PTK inhibitors reported (TKI) 1996 c-kit gof leukemia mutants 1992 CGP57148 PDGFR/ Kit/Abl TKI (Novartis) 1998 c-kit gof GIST mutants 2000 STI571/Abl structure 1998 STI571 in CML patients 2000 STI571 in GIST patients 2001 NEJM papers reporting STI571 efficacy in CML and GIST Gleevec TM approved by the FDA May 10, 2001

22 1845 CML Bennett Birchow 1960 Chr 22Δ Ph Chr Nowell Hungerford 1970 Ab-MuLV Abelson/Rabstein 1973 t(9:22) translocation Rowley 1978 v-abl protein Witte/Baltimore Reynolds/Stephenson 1982 Bcr-Abl (22:9) de Klein Stephenson Groffen Grosveld 1980 v-abl tyrosine kinase Witte/Baltimore 1984 Bcr-Abl PTK activity Konopka Witte 1990 Bcr-Abl causes CML in mice Daley/Baltimore Adams/Cory Heisterkamp/ Groffen Rosenberg/Witte 1996 STI571 inhibits CML cell growth and Bcr-Abl tumors Druker Buchdunger Lydon 2000 STI571/Abl structure Kuriyan 1911 RSV Rous 1970 v-src gene Duesberg Vogt 1952 polyoma virus Gross Eddy/Stewart 1927 W mutant mouse De Aberle 1975 c-src gene Bishop Stehelin Varmus 1977 v-src protein Brugge Erikson 1977 mt Antigen Ito/Dulbecco 1986 HZ4-FeSV/v-Kit Besmer PTK Hardy Zuckerman 1978 v-src PK Collett Erikson Bishop Varmus 1979 mt associated PTK activity Hunter/Eckhart 1987 c-kit PTK Yarden Ullrich 1979 v-src tyrosine kinase Hunter Sefton 1983 mt 1988 W is c-kit Bernstein Besmer/ Housman First PTK inhibitors (TKI) Uehara Ogawara Akiyama Graziani Umezawa Levitzki associated c-src Courtneidge/Smith 1994 c-kit gof leukemia mutants Kanakura 1992 CGP57148 PDGFR/ Kit/Abl TKI (CIBA Geigy) Lydon Matter Fabbro Buchdunger Zimmermann 1998 c-kit gof GIST mutants Kitamura 1998 STI571 in CML patients Druker Sawyers 2000 STI571 in GIST patients Demetri 2001 NEJM papers reporting STI571 efficacy in CML and GIST Druker Sawyers Demetri