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Recurrent PPP2R1A Mutations in Uterine Cancer Act Through A Published OnlineFirst August 2, 2016; DOI: 10.1158/0008-5472.CAN-15-3342 Cancer Molecular and Cellular Pathobiology Research Recurrent PPP2R1A Mutations in Uterine Cancer Act through a Dominant-Negative Mechanism to Promote Malignant Cell Growth Dorien Haesen1, Layka Abbasi Asbagh2, Rita Derua1, Antoine Hubert1, Stefanie Schrauwen3, Yana Hoorne1,Fred eric Amant3, Etienne Waelkens1, Anna Sablina2, and Veerle Janssens1 Abstract Somatic missense mutations in the Ser/Thr protein phospha- inhibitor TIPRL1. Dominant-negative Aa mutants retain bind- tase 2A (PP2A) Aa scaffold subunit gene PPP2R1A are among ing to specific subunits of the B56/B0 family and form substrate the few genomic alterations that occur frequently in serous trapping complexes with impaired phosphatase activity via endometrial carcinoma (EC) and carcinosarcoma, two clinically increased recruitment of TIPRL1. Accordingly, overexpression aggressive subtypes of uterine cancer with few therapeutic of the Aa mutants in EC cells harboring wild-type PPP2R1A options. Previous studies reported that cancer-associated Aa increased anchorage-independent growth and tumor formation, mutants exhibit defects in binding to other PP2A subunits and and triggered hyperphosphorylation of oncogenic PP2A-B56/B0 contribute to cancer development by a mechanism of haploin- substrates in the GSK3b, Akt, and mTOR/p70S6K signaling sufficiency. Here we report on the functional significance of the pathways. TIPRL1 silencing restored GSK3b phosphorylation most recurrent PPP2R1A mutations in human EC, which cluster and rescued the EC cell growth advantage. Our results reveal in Aa HEAT repeats 5 and 7. Beyond predicted loss-of-function how PPP2R1A mutations affect PP2A function and oncogenic effects on the formation of a subset of PP2A holoenzymes, we signaling, illuminating the genetic basis for serous EC develop- discovered that Aa mutants behave in a dominant-negative ment and its potential control by rationally targeted therapies. manner due to gain-of-function interactions with the PP2A Cancer Res; 76(19); 1–13. Ó2016 AACR. Introduction refs. 2–9). PPP2R1A aberrations occur early during progression in the precursor lesions (3), are distinctive for the serous Althoughnotascommonasendometrioid carcinoma (type subtype (6), and clearly distinguish uterine serous carcinomas I), serous uterine carcinoma (type II) is a highly aggressive from the clinicopathological similar ovarian high-grade serous disease characterized by high mortality due to a tendency for carcinomas (1, 7, 8). In the highly aggressive uterine carcino- early metastasis and resistance to chemotherapy (1). While sarcomas and undifferentiated carcinomas, PPP2R1A mutation genome-wide molecular changes in low grade endometrioid is also frequent (Supplementary Table S1). PPP2R1A encodes carcinomas have been revealed through The Cancer Genome the Aa subunit of type 2A protein phosphatases (PP2A), Atlas (2), the molecular changes associated with serous endo- suggesting a prominent, but so far unexplored, role for PP2A metrial cancer (EC) pathogenesis have only recently started to in the etiology of these cancers. emerge. Besides TP53 mutation (in 80%–95% of cases), rela- PP2A phosphatases are well-recognized human tumor sup- tively few additional molecular genetic aberrations can be pressors (10). They consist of a catalytic C subunit (PP2Ac), a found in these cancers, the most prevalent being alterations structural A subunit and one of multiple regulatory B-type in PPP2R1A, PIK3CA, FBXW7, CCNE1, and CHD4 (3–5). subunits defining substrate specificity of the holoenzyme Specifically, PPP2R1A is mutated in 18.4% to 43.2% of (11). Because different isoforms exist of A, B, and C subunits, all cases, depending on the study (Supplementary Table S1; their combination results in numerous PP2A holoenzymes, each with different signaling functions in a wide variety of physiological processes (12). The A subunit forms the flexible 1Laboratory of Protein Phosphorylation and Proteomics, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 2VIB scaffold between the C and B-type subunits, and is composed of Center for the Biology of Disease, Department of Human Genetics, KU 15 HEAT (Huntington, elongation factor 3, A, TOR) motifs 3 Leuven, Leuven, Belgium. Laboratory of Gynaecological Oncology, (13). Biochemical and structural studies have demonstrated Department of Oncology, KU Leuven, Leuven, Belgium. that interaction with a regulatory B-type subunit is mediated Note: Supplementary data for this article are available at Cancer Research by HEAT repeats (HR) 1–8, while interaction with C occurs Online (http://cancerres.aacrjournals.org/). through HR 11–15 (14–18). Despite these different interaction Corresponding Author: Veerle Janssens, University of Leuven (KU Leuven), domains, C and B subunit binding to the A subunit does Gasthuisberg O&N1, Herestraat 49, PO-box 901, B-3000 Leuven, Belgium. Phone: not occur independently from each other (19). For instance, 32-16-330-684; Fax: 32-16-330-735; E-mail: [email protected] N-terminal deletions of Aa inhibit C binding (14), and B55/Ba doi: 10.1158/0008-5472.CAN-15-3342 and B56/B0g3 do not bind to a C-terminally truncated Aa Ó2016 American Association for Cancer Research. (15, 20), while PR72/B" does (20). This suggests cooperativity www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 2, 2016; DOI: 10.1158/0008-5472.CAN-15-3342 Haesen et al. between specific B-type subunits and C in binding the A Cell lines subunit, while other B-type subunits may bind A independently HEK293, HEK293T, and HEC-1-A (ATCC), characterized by from C (21). Particularly the conserved C-terminal tail of the C Short Tandem Repeat profiling, were used at low passage number subunit provides additional, stabilizing contacts with B55/B (<15) immediately after receipt or after resuscitation from early and most B56/B0 subunits, but not B56/B0d,PR72/B"orstriatin/ made stocks. B"', to promote holoenzyme assembly (22). Thus, the deter- minants governing PP2A trimer assembly are significantly Lentiviral transduction dependent on the B-type subunit that is incorporated. HEK293T cells were transfected with WT/mutant Aa cloned While some PP2A complexes may be proto-oncogenic into lentiviral pLA CMV N-Flag, or with empty vector alone, in (23–25), most PP2A trimers suppress oncogenic signaling the presence of pCMV-deltaR8.91 and pMD.G-VSVG, using events (12, 26, 27). Hence, inhibition of PP2A—in the pres- Turbofect (Thermo Scientific). Lentiviral TIPRL1 shRNAs or GFP ence of oncogenic RasV12—is an absolute requirement to shRNA control (Supplementary Table S2), originally cloned in achieve full transformation of human epithelial cells immor- pLKO.1-puro (TRC 1/1.5 human shRNA library), were generated talized by expression of telomerase and SV40 large T (26–29). similarly. Twenty-four hours later, the supernatant was used to There is increasing evidence of PP2A deregulation in human transduce HEC-1-A EC cells. solid cancers and hematologic malignancies (30–32). Partic- ularly, cancer-associated missense mutations have been re- PP2A subunit binding assays ported in PPP2R1A and PPP2R1B, encoding the nonredundant HEK293 cells were transfected with appropriate PP2A sub- Aa and Ab subunits. Inactivating Ab mutations occur with unit expression vectors using PEI. 48 hours post-transfection, high frequency (up to 15%) in lung and colon cancers (33), lysates were prepared in NET buffer, and GST pulldowns, FLAG while Aa mutations have less frequently (up to 7%) been pulldowns, HA pulldowns or GFP-trapping were executed as reported in melanoma, lung and breast carcinoma (34). Most described (40). Bound proteins were boiled in 2x NuPage sample Ab and all Aa mutations affect PP2A holoenzyme formation, buffer (Invitrogen), and analyzed by immunoblotting on 4% either by interrupting interaction between A and C subunits, to 12% gels (BioRad). Membranes were blocked in 5% milk in between A and all or specific B-type subunits, or both (26, 28, TBS/0.1% Tween-20, developed with primary antibodies (Sup- 35, 36). For the characterized Aa mutants E64D/G and plementary Table S3), horseradish peroxidase-coupled secondary R418W, these binding defects result in functional haploinsuf- antibodies (DAKO) and chemiluminescence (Westernbright ficiency that promotes transformation via activation of ECL-HRP, Isogen Life Science). Densitometric quantification was the PI3K/Akt pathway (28). Accordingly, heterozygous Aa done with ImageJ. For statistics, we applied one-way multiple E64D/G knock-in mice show increased susceptibility to ben- comparisons ANOVA to the average values of all quantified bands zopyrene-induced lung cancerogenesis (37). of a given condition on a given gel. Here, we characterized 11 PPP2R1A missense mutants, commonly observed in serous uterine carcinoma. All these "IP-on-IP" approach Aa mutations are heterozygous, cluster in HEAT repeats 5 or HEK293 cells were cotransfected with expression vectors 7, and many of them are recurrent (Supplementary Table S1), for EGFP-TEV-B56d (or B56g1) and HA-Aa (WT or mutant). also in other cancer types (38). In concordance with the Forty-eight hours later, GFP-trapping was performed, and observed genetic data, our biochemical and functional assays the trapped complexes were incubated overnight at 4Cwith revealed a dominant mechanism-of-action of these mutants 0.2 mg/mL recombinant Tobacco
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