Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0218 Cancer Tumor Biology and Immunology Research The Highly Recurrent PP2A Aa-Subunit Mutation P179R Alters Protein Structure and Impairs PP2A Enzyme Function to Promote Endometrial Tumorigenesis Sarah E. Taylor1, Caitlin M. O'Connor2, Zhizhi Wang3, Guobo Shen3, Haichi Song4, Daniel Leonard1, Jaya Sangodkar5, Corinne LaVasseur6, Stefanie Avril1,7, Steven Waggoner8, Kristine Zanotti8, Amy J. Armstrong8, Christa Nagel8, Kimberly Resnick9, Sareena Singh10, Mark W. Jackson1,7, Wenqing Xu3, Shozeb Haider4, Analisa DiFeo11,12,13, and Goutham Narla5,13 Abstract Somatic mutation of the protein phosphatase 2A (PP2A) potential, and restoration of wild-type Aa in a patient- Aa-subunit gene PPP2R1A is highly prevalent in high-grade derived P179R-mutant cell line restored enzyme function endometrial carcinoma. The structural, molecular, and biological and significantly attenuated tumorigenesis and metastasis basis by which the most recurrent endometrial carcinoma– in vivo. Furthermore, small molecule–mediated therapeutic specific mutation site P179 facilitates features of endometrial reactivation of PP2A significantly inhibited tumorigenicity carcinoma malignancy has yet to be fully determined. Here, we in vivo. These outcomes implicate PP2A functional inacti- used a series of structural, biochemical, and biological vation as a critical component of high-grade endometrial approaches to investigate the impact of the P179R missense carcinoma disease pathogenesis. Moreover, they highlight mutation on PP2A function. Enhanced sampling molecular PP2A reactivation as a potential therapeutic strategy for dynamics simulations showed that arginine-to-proline substitu- patients who harbor P179R PPP2R1A mutations. tion at the P179 residue changes the protein's stable conforma- tion profile. A crystal structure of the tumor-derived PP2A mutant Significance: This study characterizes a highly recurrent, revealed marked changes in A-subunit conformation. Binding to disease-specific PP2A PPP2R1A mutation as a driver of the PP2A catalytic subunit was significantly impaired, disrupting endometrial carcinoma and a target for novel therapeutic holoenzyme formation and enzymatic activity. Cancer cells were development. dependent on PP2A disruption for sustained tumorigenic See related commentary by Haines and Huang, p. 4009 Introduction 1Department of Pathology, Case Western Reserve University School of Medicine, Uterine cancer is the most common gynecologic malignancy in Cleveland, Ohio. 2Department of Pharmacology, Case Western Reserve Univer- the United States with approximately 60,000 women diagnosed sity School of Medicine, Cleveland, Ohio. 3Department of Biological Structure, each year (1). Although most cases of uterine endometrial carci- 4 University of Washington, Seattle, Washington. Department of Pharmaceutical noma have favorable outcomes with recurrence-free long-term and Biological Chemistry, UCL School of Pharmacy, University College London, survival, outcomes for the high-grade, treatment-refractory histo- London, United Kingdom. 5Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan. 6School of Medicine, Case logic subtypes remain an important clinical problem (2, 3). Western Reserve University, Cleveland, Ohio. 7Case Comprehensive Cancer Uterine serous endometrial carcinoma (USC) accounts for only Center, Case Western Reserve University, Cleveland, Ohio. 8Department of 10% of uterine cancer cases but a disproportionate 39% of Obstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, Ohio. deaths, with a 5-year survival of 55% (2). Uterine carcinosarcoma 9 Department of Obstetrics and Gynecology, MetroHealth, Cleveland, Ohio. (UCS), a more aggressive mixed histologic subtype, is rare, <5%, 10Department of Obstetrics and Gynecology, Aultman Hospital, Canton, Ohio. > 11 but accounts for 15% of deaths, with a 5-year survival around Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, – Michigan. 12Department of Pathology, University of Michigan, Ann Arbor, Michi- 35% (4 6). Importantly, despite an overall decline in cancer- gan. 13Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan. related deaths in the United States, mortality rates for uterine cancers continue to rise (1). A lack of established disease driving Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). mechanisms for high-grade subtypes has limited the use of targeted treatment strategies, an approach that has had some Corresponding Author: Goutham Narla, University of Michigan, 32299 S Wood- success in the treatment of other cancers. New insight and vali- land Road, Ann Arbor, MI 48106. Phone: 216-368-3111; Fax: 216-368-2968; E-mail: [email protected] dation of biological drivers of disease progression are therefore needed to improve disease management. Large-scale genomic Cancer Res 2019;79:4242–57 profiling efforts have made substantial progress in identifying doi: 10.1158/0008-5472.CAN-19-0218 alterations characteristic for USC and UCS (7–10). Foremost, Ó2019 American Association for Cancer Research. both are typified by TP53 mutation, which is present in 80% to 4242 Cancer Res; 79(16) August 15, 2019 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0218 The PPP2R1A P179R Mutation Drives Endometrial Tumorigenesis 90% of cases. In addition to TP53, PPP2R1A mutation was also tissue pieces using the AllPrep DNA/RNA Mini Kit (Qiagen) in more frequent in high-grade subtypes, occurring in approximately combination with a QIAshredder (Qiagen) for initial tissue 30% of USC or UCS patients versus approximately 5% in patients homogenization. The DNA region of interest was amplified by with the endometrioid subtype (UEC). PPP2R1A encodes the PCR (PCR Master Mix, Promega) and submitted for Sanger scaffold subunit of the protein phosphatase 2A (PP2A) tumor- sequencing. Results were viewed using the software program suppressive phosphatase. 4Peaks. DNA and RNA were stored at À20C and À80C, PP2A is a serine/threonine phosphatase that is involved in the respectively. physiologic regulation of diverse signaling pathways. PP2A is also a tumor suppressor whose diminished activity contributes to Cell lines and culture conditions transformation and tumor development (11–13). Although a The UT42, UT89, UT150, and UT185 cell lines were generated number of mechanisms can underlie diminished PP2A activity by Dr. Analisa DiFeo. These stable lines were derived from primary in cancer, notable is the occurrence of "hotspot" mutations to human endometrial cancer tissue following protocols described PPP2R1A, the gene encoding the PP2A Aa-subunit. The PP2A previously (20). HEK293T cells were purchased from the ATCC. holoenzyme is a heterotrimer composed of a scaffolding A- All lines were cultured in DMEM (Corning, 10-013) supplemen- subunit (PP2A-A), catalytic C-subunit (PP2A-C), and regulatory ted with 10% FBS (GE Healthcare, SH30070.03) and 1% peni- B-subunit (PP2A-B). Each is a subunit family composed of mul- cillin–streptomycin (GE Healthcare, SV30010). Cells were main- tiple isoforms; for PP2A-A, this includes a and b isoforms. tained in humidified sterile incubator conditions with 5% CO2 at The biogenesis of canonical PP2A heterotrimers is sequential: 37C. Cells are passaged once 70% to 90% confluency is reached, first a C-subunit binds to an A-subunit and is followed by and are maintained in culture for no more than 20 passages. incorporation of one of 15 identified B-subunits (14, 15). Several Freshly thawed cells are passaged 2 to 3 times before use in activation steps must also take place, facilitated by PP2A regula- experiments. Mycoplasma testing of cell lines is performed once tory proteins, to convert the C-subunit into its catalytically active annually using a detection kit (Lonza, LT07-710), and positive cell form and to modulate the binding of B-subunits (15). Critically, lines are discarded. Authentication of cell lines was performed via throughout this process, the A-subunit serves as a highly flexible short tandem repeat profiling after the cell line was established scaffolding protein to facilitate protein interactions and trimer- (typically after two passages in culture), and compared with that ization. Cancer-derived hotspot mutations of PPP2R1A cluster at of the primary tumor. the structural interface between A- and B-subunits, and have been shown to disrupt subunit binding to varying degrees (11, 16–18). Generation of stable cell lines A recent report characterizing several hotspot mutations found Standard methodologies were used; details are provided in the that these mutant proteins have increased binding to the PP2A Supplementary Methods. inhibitor TIPRL, resulting in a dominant-negative phenotype (18). Our data suggest this mechanism may be true for some hotspot Immunoblotting mutations but may not hold true for others that are most specific Standard methodologies were used; details and the primary and recurrent in high-grade endometrial carcinoma. Specifically, antibodies used are provided in the Supplementary Methods. although PPP2R1A mutations are found in cancers of multiple origins, they are striking for USC and UCS not only for their high Real-time PCR prevalence, but also for the nearly exclusive occurrence of muta- Standard