Published OnlineFirst October 24, 2018; DOI: 10.1158/1541-7786.MCR-18-0777 Oncogenes and Tumor Suppressors Molecular Cancer Research PTPN11 Plays Oncogenic Roles and Is a Therapeutic Target for BRAF Wild-Type Melanomas Kristen S. Hill1, Evan R. Roberts1, Xue Wang1, Ellen Marin1, Taeeun D. Park1, Sorany Son1, Yuan Ren1, Bin Fang2, Sean Yoder3, Sungjune Kim4,5, Lixin Wan1, Amod A. Sarnaik6, John M. Koomen1,2, Jane L. Messina6,7, Jamie K. Teer8, Youngchul Kim8, Jie Wu9, Charles E. Chalfant10,11, and Minjung Kim1,6,10 Abstract Melanoma is one of the most highly mutated cancer types. noma tumorigenesis. Melanoma cells derived from this mouse To identify functional drivers of melanoma, we searched for model showed doxycycline-dependent tumor growth in nude cross-species conserved mutations utilizing a mouse melano- mice. Silencing PTPN11E76K expression by doxycycline with- ma model driven by loss of PTEN and CDKN2A, and identified drawal caused regression of established tumors by induction mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes of apoptosis and senescence, and suppression of proliferation. the SHP2 protein tyrosine phosphatase that activates the Moreover, the PTPN11 inhibitor (SHP099) also caused regres- Q61K RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene sion of NRAS -mutant melanoma. Using a quantitative in leukemia, lung, and breast cancers, its roles in melanoma are tyrosine phosphoproteomics approach, we identified GSK3a/ not clear. In this study, we found that PTPN11 is frequently b as one of the key substrates that were differentially tyrosine- activated in human melanoma specimens and cell lines and is phosphorylated in these experiments modulating PTPN11. required for full RAS/RAF/MAPK signaling activation in BRAF This study demonstrates that PTPN11 plays oncogenic roles in wild-type (either NRAS mutant or wild-type) melanoma cells. melanoma and regulates RAS and GSK3b signaling pathways. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. Implications: This study identifies PTPN11 as an oncogenic In Pten- and Cdkn2a-null mice, tet-inducible and melanocyte- driver and a novel and actionable therapeutic target for BRAF specific PTPN11E76K expression significantly enhanced mela- wild-type melanoma. Introduction cross-species conserved genomic alterations, which led to the identification of cIAP and YAP1 as driver oncogenes in liver cancer Genetically engineered mouse models have been utilized to (1) and NEDD9 in melanoma (2). Whole-genome sequencing mine complex human genomic data to identify novel human analysis of a tumor developed in an acute promyelocytic leukemia cancer genes. Integration of high-resolution copy-number profiles (APL) mouse model uncovered recurrent somatic mutations in generated using mouse and human tumor genomes identified Jak1, pointing to the importance of the JAK/STAT pathway in pathogenesis of human APL (3). These studies have demonstrated that mice and humans sustain syntenic genetic events in the 1Department of Molecular Oncology, Moffitt Cancer Center, Tampa, Florida. development of cancer, that mouse–human comparative onco- 2Department of Proteomics, Moffitt Cancer Center, Tampa, Florida. 3Molecular genomics can facilitate the discovery of new cancer genes that were 4 Genomics Core, Moffitt Cancer Center, Tampa, Florida. Department of not immediately obvious from the human cancer genome alone, fi 5 Immunology, Mof tt Cancer Center, Tampa, Florida. Department of Radiology, and that the mouse models may be utilized to gain insights into Moffitt Cancer Center, Tampa, Florida. 6Department of Cutaneous Oncology, Moffitt Cancer Center, Tampa, Florida. 7Department of Pathology, Moffitt the function of these new cancer genes. Cancer Center, Tampa, Florida. 8Department of Biostatistics and Bioinformatics, Melanoma displays frequent activation of RAS/RAF/MAPK Moffitt Cancer Center, Tampa, Florida. 9Peggy and Charles Stephenson Cancer and PI3K/AKT signaling pathways, as well as inactivation of Center, University of Oklahoma Health Sciences Center, Oklahoma City, PTEN and CDKN2A (INK4A/ARF) tumor suppressors via genetic 10 Oklahoma. Department of Cell Biology, Microbiology, and Molecular Biology, and epigenetic alterations. The high frequency of activating muta- 11 University of South Florida, Tampa, Florida. Department of Research Service, tions in BRAF (40%–60%) and NRAS (15%–25%) support James A. Haley Veterans Hospital, Tampa, Florida. the importance of the RAS/RAF/MEK/ERK pathway in melanoma Note: Supplementary data for this article are available at Molecular Cancer (4, 5). Several recent genomic studies have identified mutations in Research Online (http://mcr.aacrjournals.org/). genes regulating the RAS pathway such as RasGAPs (NF1, RASA1, Corresponding Author: Minjung Kim, University of South Florida, 4202 East RASA2) and PTPN11 in melanoma (4, 6–10). These Ras regulators Fowler Ave., ISA2015, Tampa, FL 33620. Phone: 813-600-9441; Fax: 813-905- have the potential of being exploited as therapeutic targets. 9919; E-mail: [email protected] Protein-Tyrosine Phosphatase (PTP), Non-Receptor Type 11 doi: 10.1158/1541-7786.MCR-18-0777 (PTPN11, also known as SHP2) is ubiquitously expressed in Ó2018 American Association for Cancer Research. various tissues and cell types, and activates the Ras signaling www.aacrjournals.org 583 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 24, 2018; DOI: 10.1158/1541-7786.MCR-18-0777 Hill et al. pathway as downstream of most, if not all, receptor tyrosine 5037, when tumors reached 200 mm3, mice were randomly kinases (RTK). PTPN11 contains two SH2 domains and a PTP enrolled onto vehicle control (1% carboxymethyl cellulose/ domain. PTPN11 is autoinhibited via a SH2–PTP domain inter- 0.5% Tween-80), MEK162 (25 mg/kg, p.o., b.i.d.), or SHP099 action and is activated by binding to tyrosine-phosphorylated (100 mg/kg, p.o., qd) treatment groups. All animals were RTKs, immune-inhibitory receptors, or docking proteins (such as maintained according to the guidelines of the Comparative Gab1/Gab2, IRS, FRS) and by point mutations that disrupt Medicine Department of the University of South Florida the SH2–PTP domain interaction. RTKs, such as EPHA2, (M4473 and R3312). phosphorylate Y542 and Y580 on PTPN11, which prolongs ERK activation by maintaining PTPN110s open conformation Whole-exome sequencing analysis of mouse melanomas with (11). Mutations in PTPN11 have been linked to Noonan and matched normal tissue LEOPARD syndromes and pathogenesis of multiple cancer types. Genomic DNA from 3 melanomas developed in IP mice with Oncogenic roles of PTPN11 in leukemia, lung, and breast matched normal tissue (kidney or tail) was subjected to whole- cancers have been established with PTPN11 regulating invasion, exome capture and paired-end sequencing on Illumina Hi-Seq metastasis, apoptosis, senescence, DNA damage, cell prolifera- 2000 sequencer by Beckman Coulter Genomics, Inc. Overall tion, cell-cycle progression, and drug resistance (12, 13). How- average coverage of 63.5X was achieved. Sequence reads obtained ever, PTPN11 has tumor-suppressive roles in liver cancer (14) and were aligned to the mm10 mouse reference sequence (C57BL/6J in bone/cartilage cancer (15), supporting PTPN110s cell context– strain) using the Burrows–Wheeler Alignment tool (18). Known dependent effects. SNPs of FVB/N mouse strain (strain background of the IP model) PTPN11 PTP activity regulates several molecules involved in from v.3 of the Mouse Genomes Project (19) and variants mapped Ras signaling (16). Specifically, PTPN11 negatively regulates outside of targeted region were removed. Utilizing Strelka (20), RasGAP recruitment by dephosphorylating RTKs (e.g., EGFR and somatic single-nucleotide variants (SNV) and small indels PDGFRb), leading to activation of Ras signaling. PTPN11 also were identified from the aligned sequencing reads of matched dephosphorylates PAG (phosphoprotein associated with glyco- tumor-normal samples. sphingolipid-enriched membrane microdomains) and paxillin, resulting in the release of CSK (C-terminal Src kinase) and Phosphoproteomic analysis subsequent activation of Src family kinases (SFK). A recent study Tumors were homogenized in denaturing buffer containing by Bunda and colleagues showed dephosphorylation and activa- 8 mol/L urea and clarified by centrifugation. Proteins were tion of Ras by PTPN11 (17). Additional targets of PTPN11 include reduced, alkylated, and digested overnight with trypsin Sprouty, signal regulatory protein-a, and protein zero-related. (Worthington). After buffer exchange, tyrosine-phosphorylated Although the list of PTPN11 substrates continues to grow, it is peptides were immunoprecipitated using anti-phosphotyrosine possible that PTPN11 dephosphorylates differential sets of sub- antibody beads (p-Tyr-100), followed by analysis on LC-MS/MS strates depending on the cell context in each cancer type. peptide sequencing (RSLC-QExactive Plus, Thermo), as previous- In this study, to identify functional driver mutations of mel- ly described (21). Label-free quantification by MaxQuant (22) anoma, we analyzed melanoma genomes from a mouse model produced 245 molecularly defined tyrosine phosphorylation sites driven by loss of CDKN2A (INK4A/ARF) and PTEN (IP model), with relative quantification for the levels of phosphorylation. commonly observed alterations in human melanoma patients, LIMMA analysis was then performed to identify