Gynecological malignancies (ovarian clear cell and endometrioid carcinomas, endometrial serous, endometrioid and clear cell carcinomas) harbor somatic mutations in the gene PPP2R1A, the scaffolding subunit of the serine/threonine protein phosphatase 2A (PP2A) complex. The PP2A complex is composed of an A (scaffold), C (catalytic) subunit, and different B (regulatory) subunits. The PPP2R1A mutations are proposed to play a role in PP2A B-subunit binding, and thus formation of a functional PP2A phosphatase complex. The aim of this study is to determine how PPP2R1A mutations affect the binding of B-subunits and other novel interactions within the context of ovarian and endometrial carcinomas.

The ovarian clear cell line RMG2 and endometrial carcinoma cell lines Hec-1-A and Hec50 all harbor PPP2R1A mutations. The mismatch repair deficient cell line Hec-1-A express a PPP2R1A W257L heterozygous mutation; therefore somatic cell knockout technology was used to generate isogenic PPP2R1A clones that express only mutant PPP2R1A, and clones that express more wild-type than mutant. These Hec-1-A isogenic cell lines were used for PPP2R1A co-immunoprecipiation (Co-IP) followed by MS/MS-SWATHTM analysis to identify PP2A B-subunits and other novel protein interactions.

Our preliminary results using Hec-1-A isogenic cell lines suggest that some PP2A B-subunits (PPP2R5D and PPP2R2A) are found to be decreased in abundance in the mutant cell lines. We were able to identify the catalytic subunit (PPP2CA) in PPP2R1A Co-IPs in all cell lines. We also show that PPP2R1A interacts with novel proteins such as histones (Histone 1, Histone H2B type 1-N), ubiquitin ligases (TRIM21) and proteins involved in mitotic control (ANKLE2). Of particular interest we have identified the cancer gene nucleophosmin (NPM1) as a potential novel interactor of PP2A.

We have established the isogenic Hec-1-A cell lines as a model system to determine the functional effects of a somatic PPP2R1A mutation. These cell lines have shown that the PPP2R1A mutation affects the ability of some B-subunits to interact and form the PP2A complex. We have also shown that PP2A may interact with novel proteins such as NPM1 and histones. Ongoing work will be completed to determine how these interactions function in the context of ovarian and endometrial carcinomas.

Citation Format: Melissa K. McConechy, Vincent Chen, Gregg Morin, James Brenton, David G. Huntsman. PPP2R1A mutations affect PP2A complex formation, and novel protein interactions in gynecological carcinoma cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A25.