Targeting Cell Survival Signaling for Therapeutic Discovery 公开
Kasinski, Andrea Lynn (2009)
Published
Abstract
Abstract
Targeting Cell Survival Signaling for Therapeutic Discovery
By: Andrea Lynn Kasinski
The completion of the human genome sequence has greatly facilitated gene profiling to reveal tumor specific signatures and the identification of tumor-addicted oncogenes and pathways. These advances offer unprecedented opportunities for anticancer therapeutic discovery and development. The emerged cancer survival signaling pathways guide our characterization of promising therapeutic agents while lead us to novel strategies for targeting these tumor-addicted regulatory mechanisms. By taking advantage of these developments, my dissertation seeks (i) to understand the mechanism of action of a natural product analog, EF24, for anticancer therapeutic discovery and (ii) to unravel the transcriptional control machinery of a specific member of the 14-3-3 protein family, a master regulator of cell survival signaling, for future therapeutic interventions.
Curcumin, a natural product, has shown numerous therapeutic benefits but suffers from low potency and poor absorption. To overcome these limitations, a structural analog, EF24, was developed and exhibited a potent anticancer activity. However, its molecular target remains to be defined. My research describes a mechanism by which EF24 potently suppresses the NF-kB signaling pathway through direct action on the I-kB kinase (IKK): (i) EF24 rapidly blocks the nuclear translocation of NF-kB with a potency ten times better than that of curcumin, (ii) EF24 effectively inhibits TNFa-induced I-kB phosphorylation and degradation, and (iii) EF24 directly inhibits the catalytic activity of IKKb in an in vitro reconstituted system. This study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNFa-induced NF-kB signaling by EF24 extends the therapeutic application of EF24 to other NF-kB-dependent diseases besides cancer, including various inflammatory diseases.
Dysregulated 14-3-3 proteins have been associated with poor survival of certain cancer populations. Targeting 14-3-3-mediated survival signaling has emerged as a promising anticancer strategy. However, the high level of sequence similarity among its seven isoforms and functional redundancy pose significant challenges. My research aims at defining the transcriptional mechanism that specifically controls the expression of a highly oncogenic isoform, 14-3-3z ( ywhaz ). Through a series of molecular and genetic approaches, my research identified five transcript variants of ywhaz , determined the importance of ywhaz variant 1c, revealed a functional CRE element within its promoter, and discovered two CREB family members as ywhaz transcription factors. Indeed, silencing of ATF-1, a CREB member, resulted in reductions in two of the five variants accompanied by significantly decreased 14-3-3z protein. This study defines the first transcriptional regulatory system that directs the expression of an oncogenic 14-3-3 isoform and reveals a mechanism by which ATF-1-mediated pathways control the expression and function of 14-3-3z and 14-3-3z-regulated signaling network.
Table of Contents
Table of Contents
Chapter 1: Introduction.................................................................................................... 1
Curcumin........................................................................................................................ 3
Curcumin's therapeutic potential.................................................................................. 4
Curcumin's use as a chemopreventive agent ................................................................ 4
Curcumin's use as a chemotherapeutic agent .............................................................. 5
Curcumin's therapeutic pitfalls..................................................................................... 6
Curcumin exhibits poor bioavailability......................................................................... 6
Techniques to improve upon the bioavailability of curcumin....................................... 8
Blocking glucuronidation ............................................................................................. 8
Generation of curcumin analogs to improve its pharmacological properties ............... 8
NF-kB............................................................................................................................ 11
Introduction and identification of NF-kB................................................................... 11
NF-kB in immune response and its tie to tumorigenesis............................................. 14
Elevations in NF-kB activity lead to aberrant T-cell activation ................................... 14
Elevations in NF-kB activity lead to induction of Cox-2 ............................................. 17
Elevations in NF-kB activity lead to chemoresistance and radioresistance ................ 19
Signaling through NF-kB............................................................................................ 19
Alternative (non-canonical) NF-kB signaling ............................................................. 20
Classical NF-kB signaling .......................................................................................... 20
Targeting NF-kB pro-survival signaling for therapeutic development......................... 24
Targeting NF-kB through IKK inactivation ................................................................ 24
14-3-3 family of proteins.............................................................................................. 26
Identification of 14-3-3 proteins................................................................................ 26
Genomic organization and species conservation of 14-3-3 isoforms.......................... 26
Conservation of 14-3-3 between isoforms and species ............................................... 29
14-3-3 Regulation....................................................................................................... 29
Post-translational Regulation of 14-3-3 monomers .................................................... 30
Phosphorylation dependent (and independent) regulation of 14-3-3 binding to client proteins 30
Recognition of phosphorylated clients by 14-3-3 proteins .......................................... 30
Outcome of 14-3-3 binding to client proteins............................................................. 31
14-3-3 binding changes the localization of a protein ................................................. 33
14-3-3 binding can alter enzymatic function of the client ........................................... 33
14-3-3 binding contributes to altered protein-protein interactions ............................. 34
Cellular survival functions associated with 14-3-3..................................................... 35
Transcriptional and translational regulation of 14-3-3 proteins................................... 37
Role of 14-3-3 in disease........................................................................................... 38
Targeting 14-3-3 for therapeutic development............................................................ 40
CREB/ATF family of transcription factors................................................................. 41
Identification.............................................................................................................. 41
Protein structure......................................................................................................... 41
Signaling to CREB and ATF Family Members............................................................ 44
Functions downstream of CREB................................................................................ 44
CREB family members in oncogenesis....................................................................... 45
Chapter 2: Inhibition of IKK-NF-kB signaling pathway by EF24, a novel monoketone analogue of curcumin........................................................................................................................................... 47
Abstract......................................................................................................................... 49
Introduction.................................................................................................................. 50
Materials and Methods................................................................................................. 53
Results........................................................................................................................... 58
EF24 exhibits a more potent cytotoxic effect than curcumin...................................... 58
High content analysis (HCA) revealed an effective EF24 action in blocking nuclear translocation of NF-kB 58
EF24 inhibits TNFa-induced I-kB phosphorylation and subsequent degradation........ 60
EF24 directly inhibits the IKKb kinase activity.......................................................... 61
Discussion...................................................................................................................... 64
Reference...................................................................................................................... 68
Footnotes....................................................................................................................... 71
Figure legends............................................................................................................... 72
Chapter 3: Transcriptional regulation of ywhaz, the gene encoding 14-3-3z............. 86
Introduction.................................................................................................................. 89
Materials and Methods................................................................................................. 92
Results......................................................................................................................... 102
14-3-3 zeta is encoded from at least five different transcript variants...................... 102
ywhaz variant 1c is the most dominant variant identified......................................... 103
Identification of a CRE regulatory region in the proximal promoter for variant 1c... 105
A putative CRE element is necessary for basal activation from the 14-3-3 promoter 106
Nuclear proteins bind the CRE element in vitro........................................................ 106
ATF-1 and CREB bind to the CRE element in vitro .................................................. 108
ATF-1 is recruited to the endogenous ywhaz promoter after stimulation with TNF-a 109
Silencing of ATF-1 decreased 14-3-3 zeta expression.............................................. 110
ATF-1 regulates expression of ywhaz transcript variants 1c and 1e.......................... 111
Discussion.................................................................................................................... 113
References................................................................................................................... 116
Figure Legends............................................................................................................ 118
Chapter 4: Discussion.................................................................................................... 137
Chapter 5: References................................................................................................... 143
Appendix: Identification of genes involved in 14-3-3-dependent rapamycin signaling 157
Abstract....................................................................................................................... 158
Background and Significance..................................................................................... 158
mTOR controls cell growth signaling in normal and cancer cells ............................ 158
Rapamycin and its analogs have shown promising anticancer properties through inhibition of mTOR 159
14-3-3 proteins contribute to rapamycin resistance ................................................. 159
Introduction................................................................................................................ 160
Materials and Methods............................................................................................... 162
Results......................................................................................................................... 163
Discussion.................................................................................................................... 164
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