Investigating the Tumor Suppressive Role of PP2Ac Methylation: Implications for SV40- and Polyomavirus-mediated Transformation Público
Jackson, Jennifer Beverly (2012)
Abstract
PP2A regulates growth and survival pathways and its dysfunction has been linked to cancer. The heterotrimeric PP2A holoenzyme consists of catalytic C (PP2Ac), structural A, and regulatory B-type subunits. Reversible methylation of PP2Ac by LCMT-1 and PME-1 differentially regulates the binding of certain B-type subunits and thus PP2A function. In addition to the cellular B-type subunits, some DNA tumor virus oncoproteins including polyomavirus small (PyST) and middle (PyMT) tumor antigens and SV40 small tumor antigen (SVST) function as viral B-type subunits. These viral oncoproteins alter PP2A function and promote transformation in part by replacing certain cellular B-type subunits. Interestingly, while the B-type subunits replaced by these oncoproteins appear to exhibit a binding preference for A/C dimers containing methylated PP2Ac (methylation-sensitive), the only tumor antigen tested to date, PyMT, does not (methylation-insensitive). In Chapter 3.1, I hypothesized that circumventing the cellular control of PP2A by PP2Ac methylation is a general strategy for MT- and ST-mediated transformation. Results indicated that SVST and PyST also bind to PP2A in a methylation-insensitive manner. Furthermore, reduction of PP2Ac methylation enhanced transformation through activation of the Akt and S6K1 pathways. These results support the hypothesis that replacing methylation-sensitive B-type subunits with methylation-insensitive B-type subunits, thereby circumventing control of PP2A by methylation, is a general strategy for MT and ST-mediated transformation. In Chapter 3.2, I tested the individual contributions to transformation of the N-terminal, PP2A-independent portion of SVST and of reducing methylation-sensitive PP2A complexes in conjunction with B'γ knockdown. The findings revealed that MT- and ST-mediated transformation is likely modulated through the concomitant expression of the N-terminal domain and reduction of multiple methylation-sensitive PP2A complexes. Finally, in Chapter 3.3, I found that reducing PME-1 could reduce transformation. Overall, these studies provide insights into a novel strategy that T antigens may utilize to circumvent cellular regulation of PP2A by methylation. The research here also provides evidence for the involvement of PP2Ac methylation in supporting the tumor suppressive role of PP2A. Therefore, disruption of PP2Ac methylation may contribute to cancer and promoting this methylation (e.g. PME-1 inhibitors) may serve as a therapeutic target for cancer.
Table of Contents
Table of Contents
Chapter 1: GEneral Introduction __________________________________1
Protein Phosphorylation____________________________________________1Basic Structure of PP2A____________________________________________2
Reversible Methylation of PP2A C Subunit______________________________5 Role of PP2Ac Methylation__________________________________________7 PP2A Function: A Pleotrophic Protein_________________________________10Akt/PKB Pathway________________________________________________12
Akt-mTOR-S6 Kinase Pathway______________________________________14 ERK/MAPK Pathway______________________________________________17 Cell Cycle______________________________________________________18 Apoptosis______________________________________________________22 Cancer and PP2A________________________________________________26 PP2A: Evidence for a Tumor Suppressor______________________________27 DNA Tumor Viruses: Targeting PP2A Assembly_________________________30 Simian Virus 40 (SV40)___________________________________________31 HEKTERASB56γ: A Genetically Defined Transformation Cell Line___________34 Polyomavirus___________________________________________________37Goals of the Dissertation__________________________________________40
CHAPTER 2: METHODS AND MATERIALS___________________________44 Cell Culture_____________________________________________________44 Transfection of shRNAs for Lentivirus Production________________________44Lentiviral Titering and Infections_____________________________________45
Creation of the LCMT-1-Rescue cDNA Sequence_________________________47 Creation of HEKTERASB56γ Cells That Stably Express Akt-AA, PME-1, ST110, or an shRNA-resistant LCMT-1 ________________________________47 Antibodies_______________________________________________________48 Cell Lysis and Western Analysis______________________________________49Transfection Procedure for Expression of SVST, PyST and PP2Ac_____________50
Immunoprecipitation_______________________________________________50 PP2A C Subunit Methylation Assay____________________________________51 Anchorage-Independent Growth (Soft Agar Analysis)_____________________52 Suspension Cultures_______________________________________________53 Anchorage-Dependent Growth Assays_________________________________54 Anchorage-Dependent Death Assays__________________________________54 Time-Lapse Microscopy_____________________________________________55 CHAPTER 3: RESULTS____________________________________________56Chapter 3.1: Circumventing Cellular Control of PP2A by Methylation
Promotes Transformation in an Akt-dependent Manner____________________56SVST and PyST Do Not Require PP2Ac Methylation for PP2A
Heterotrimer Formation_____________________________________________56 Loss of LCMT-1 Promotes Transformation_______________________________57LCMT-1 Knockdown Does Not Affect Cell Size, Proliferation Rate,
Survival, or the Akt and p70/p85 S6K Signaling Pathways during Normal
Anchorage-Dependent Growth________________________________________59LCMT-1 Knockdown Activates the Akt and p70/p85 S6K Pathways
in Anchorage-Independent Conditions__________________________________60Effects of LCMT-1 Knockdown on Growth and Signaling Are Rescued
by Exogenous Expression of LCMT-1___________________________________63Akt Activation Is Necessary for the Enhanced Transformation Caused by
LCMT-1 Knockdown________________________________________________65Overexpression of the PP2Ac Methylesterase, PME-1, Causes Similar Changes
in Anchorage-Independent Growth and Signaling As LCMT-1 Knockdown______66
Chapter 3.2: Investigating the Potential Targets of SV40 Small T Antigen and
the Potential Contributions of the SV40 Small T Antigen PP2A-independent Region to Transformation___________________________________________90Expression of the PP2A-independent region of SVST does not complement
B'γ knockdown to further enhance transformation________________________90Suppression of methylation-sensitive, SVST-targeted PP2A B-type subunits
in combination with B'γ knockdown reveals differential effects on cell
growth, survival, and transformation__________________________________91 Suppressing Bα___________________________________________________92 Suppressing B'α___________________________________________________93 Suppressing Bδ___________________________________________________95Chapter 3.3: Sustained PP2Ac Methylation Enhances the Tumor Suppressive
Function of PP2A through Down-regulation of the Akt Pathway______________109
Suppression of PME-1 Reduces Transformation___________________________109PME-1 Knockdown Does Not Affect Proliferation Rate or the Akt and p70/
p85 S6K Signaling Pathways during Normal Anchorage-Dependent Growth_____111
PME-1 Knockdown Downregulates the Akt and p70/p85 S6K Pathways
in Anchorage-Independent Conditions__________________________________112 CHAPTER 4: DISCUSSION_____________________________________119 Circumventing Normal Cellular Control of PP2A by PP2Ac Methylation is a General Strategy for ST- and MT-mediated Transformation_________________119 Expression of the PP2A-independent Region of SVST Does Not Complement B'γ Knockdown in Transformation_____________________________________125Suppression of Methylation-Sensitive, SVST-targeted PP2A B-type Subunits in
Combination with B'γ Knockdown Reveals Differential Effects on Cell Growth,
Survival, and Transformation_________________________________________127 Sustained PP2Ac Methylation Enhances the Tumor Suppressive Function of PP2A through Down-regulation of the Akt Pathway________________________137 Closing Remarks: Implications for Cancer_______________________________140 Future Directions __________________________________________________143CHAPTER 5: REFERENCES_____________________________________149
List of Figures1. General Schematic of PP2A Holoenzyme Composition page 4
2. General Schematic of Reversible Methylation of PP2Ac page 6
3. General Schematic of PP2A Functions page 11
4. The HEKTER Experimental Cell Model page 35
5. General model illustrating the regulation of PP2A complexes by PP2Ac methylation and PP2A's regulation of progrowth, prosurvival pathways page 41
6. Incorporation of SVST and PyST into PP2A heterotrimers independently of
PP2Ac carboxyl methylation page 697. Knocking down LCMT-1 promotes transformation page 71
8. Adherent control and LCMT-1 knockdown lines show no difference in
growth, death, or Akt and S6K signaling page 73
9. LCMT-1 knockdown activates Akt and S6K signaling in anchorage-
independent conditions page 7510. Effects of LCMT-1 knockdown on transformation are rescued by expression
of an shRNA-resistant LCMT-1 page 7711. LCMT-1 overexpression confirms the tumor suppressive role of
PP2Ac methylation page 7912. Effects of LCMT-1 knockdown on biochemical signaling are rescued by
re-expression of LCMT-1 page 8113. Akt activation is necessary for the enhanced transformation caused by
LCMT-1 knockdown page 8314. Effects of LCMT-1 knockdown on biochemical signaling are reversed
by expression of dominant-negative Akt page 84
15. Overexpression of the PP2A methylesterase, PME-1, enhances
transformation page 8616. Overexpression of the PP2A methylesterase, PME-1, elicits similar
biochemical changes as LCMT-1 suppression page 88
17. Expression of the PP2A-independent region of SVST does not complement
B'γ knockdown to further enhance transformation page 99
18. Knocking down Bα does not significantly enhance transformation page 101
19. Knocking down B'α reduces transformation page 103
20. Knocking down Bδ in HeLa cells causes widespread death page 105
21. Bδ knockdown or SV40 small T (SVST) expression in HeLa cells causes
mitotic defects and apoptosis page 107 22. Knocking down PME-1 reduces transformation page 11423. Adherent control and PME-1 knockdown lines show no difference in growth
or Akt and S6K signaling page 11624. PME-1 knockdown downregulates the Akt and S6K pathways in anchorage-
independent conditions page 11725. Proposed model for the negative regulation of anchorage-independent growth
and survival by methylation-sensitive PP2A heterotrimers page 145 26. Proposed model for the strategies exploited by SV40 small T (SVST) to promote transformation page 147LIST OF TABLES
1. List of shRNAs (acquired from The RNAi Consortium (TRC)) targeting
PP2A B-type subunits, LCMT-1 and PME-1 page 45About this Dissertation
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