Pharmacological Modulation of PP2A Activity in Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Daniel Bachovchin, A.B. -
Award Cycle: 2010 (Cycle 16) Grant #: 16GB-0062 Award: $29,366
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2010)

Dysregulated enzymatic pathways in breast cancer cells can be exploited as biomarkers and therapeutic targets. Consequently, the discovery and biochemical understanding of these pathways remains an important, yet challenging goal for breast cancer researchers. Mounting evidence has implicated the inactivation of protein phosphatase 2A (PP2A) as a critical step malignant transformation, but the molecular mechanisms of inactivation are not yet understood. One enzyme thought to contribute to the deactivation of PP2A is protein methylesterase-1 (PME-1), a pro-tumorigenic serine hydrolase that removes an activating post-translational methylation on the C-terminus of PP2A. These results suggest that the activation of PP2A by inhibition of PME-1 may represent a novel strategy to combat breast cancer.

Previous research efforts in the Cravatt (mentor’s) laboratory have utilized a proteomics technology, called based protein profiling (ABPP), to discover the first selective PME-1 inhibitor. We are therefore in a unique position to study the enigmatic tumor suppressor PP2A through the use of the first pharmacological tool that specifically affects its regulation. In this project, we plan to use quantitative mass spectrometric methods to globally profile changes in cellular phosphorylation events in response to inhibitor treatment. In addition, we will genetically engineer breast cancer cell lines using shRNA technology to constitutively diminish PME-1 expression and perform similar analyses on these cell lines. We will then investigate the relationships of these events to breast cancer cell growth and malignancy.

In the future, we plan to examine PME-1 activity and phosphopeptide signatures in primary human breast tumors as part of our ongoing, NIH-funded collaboration with Stefanie Jeffrey at Stanford Medical School. Ultimately, we hope that this research could potentially lead to new diagnostic markers and therapeutic targets for breast cancer.




Final Report (2011)

Since the funding of this fellowship, we have made significant progress toward mapping the global phosphorylation changes in response to alteration of PP2A methylation. We have successfully made a series of cell lines with overexpression or knockdown of PME-1 and/or LCMT-1 (the methyl transferase that installs the PP2A carboxymethylation). Each of these cell lines exhibits large changes in levels of PP2A methylation.

Recently, we also discovered an extraordinarily potent and selective PME-1 inhibitor, ABL127 (discussed in detail below), which is more effective than DAB8. Upon treatment of cell lines with nanomolar concentrations, ABL127 significantly decreases in the amount of PP2A. With these new cell lines and inhibitor now in hand, we employed stable isotope labeling of amino acids in cell culture (SILAC) to differentially label proteins with stable isotopes to generate isotopically “light” and “heavy” cell lines. These lines, when pooled and analyzed by mass spectrometry, yield accurate quantification by comparing intensities of light and heavy peptide peaks. Studies comparing these cell lines +/- inhibitor employing these mass-spectrometry techniques with our cell lines and inhibitor are in progress.

In addition, ABL127 selectively inhibits PME-1 in living mice. This results in significant decrease in the levels of demethylated PP2A. This tool has now given us the first opportunity to evaluate the role that PP2A methylation plays in animal models of breast cancer pathogenesis, studies that are currently underway. In the last year, we have successfully developed an inhibitor, ABL127, that selectively inhibits PME-1 in living mice. In vivo PME-1 inhibition results in significant decrease in the levels of demethylated PP2A. This tool has now given us the first opportunity to evaluate the role that PP2A methylation plays in animal models of breast cancer pathogenesis, studies that are currently underway.



Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors.
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Bachovchin DA, Mohr JT, Speers AE, Wang C, Berlin JM et al and Cravatt B
Yr: 2011 Vol: 108 Nbr: 17 Abs: Pg:6811-6

Discovery and Optimization of Sulfonyl Acrylonitriles as Selective, Covalent Inhibitors of Protein Phosphatase Methylesterase-1.
Periodical:Journal of Medicinal Chemistry
Index Medicus: J Med Chem
Authors: Bachovchin DA, Zuhl AM, Speers AE, Wolfe MR, Weerapana E, Brown SJ, Rosen H, Cravatt BF
Yr: 2011 Vol: 54 Nbr: 14 Abs: Pg:5229-5236