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Candidate Tumor Suppressor HYAL2 Is a Glycosylphosphatidylinositol (GPI

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Candidate Tumor Suppressor HYAL2 Is a Glycosylphosphatidylinositol (GPI Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation Sharath K. Rai*†, Fuh-Mei Duh†‡, Vladimir Vigdorovich*, Alla Danilkovitch-Miagkova§, Michael I. Lerman§, and A. Dusty Miller*¶ *Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and ‡Intramural Research Support Program, Science Applications International Corporation-Frederick, §Laboratory of Immunobiology, Division of Basic Sciences, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702 Edited by John M. Coffin, Tufts University School of Medicine, Boston, MA, and approved January 29, 2001 (received for review December 4, 2000) Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung targeted to the lung, provided that the pathogenic features of the cancer, but JSRV is a simple retrovirus having no known oncogenes. virus can be controlled. Identification of the JSRV receptor and Here we show that the envelope (env) gene of JSRV has the unusual determination of the pattern of receptor expression in the lung will property that it can induce transformation in rat fibroblasts, and thus help identify cells that can be targeted by JSRV vectors. is likely to be responsible for oncogenesis in animals. Retrovirus entry To facilitate identification of the JSRV receptor, we made into cells is mediated by Env interaction with particular cell-surface retrovirus packaging cells that produce a retrovirus vector with Gag receptors, and we have used phenotypic screening of radiation hybrid and Pol proteins from Moloney murine leukemia virus, Env protein cell lines to identify the candidate lung cancer tumor suppressor from JSRV, and a retroviral vector genome (LAPSN) that encodes HYAL2͞LUCA2 as the receptor for JSRV. HYAL2 was previously de- human placental alkaline phosphatase (AP) as a marker for vector scribed as a lysosomal hyaluronidase, but we show that HYAL2 is transduction (4). This vector was able to transduce sheep and actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface human cells, with the exception of HeLa cells, but was unable to protein. Furthermore, we could not detect hyaluronidase activity transduce cells from mice, rats, and hamsters because of the lack of associated with or secreted by cells expressing HYAL2, whereas we functional receptors for JSRV Env. could easily detect such activity from cells expressing the related The presence of a functional receptor in human cells and its serum hyaluronidase HYAL1. Although the function of HYAL2 is absence in hamster cells allowed us to use phenotypic screening currently unknown, other GPI-anchored proteins are involved in of human͞hamster radiation hybrid cell lines (5) to localize the signal transduction, and some mediate mitogenic responses, suggest- receptor in the human genome. We assayed the Stanford G3 ing a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung panel of 83 human͞hamster radiation hybrid clones for suscep- cancer induced by JSRV closely resembles human bronchiolo-alveolar tibility to transduction by the JSRV vector. Each hybrid contains carcinoma, a disease that is increasing in frequency and now accounts a full complement of hamster chromosomes and multiple radi- for Ϸ25% of all lung cancer. The finding that JSRV env is oncogenic ation-fragmented pieces of human DNA with average size of Ϸ4 and the identification of HYAL2 as the JSRV receptor provide tools for megabases and representing Ϸ18% of the human genome. These further investigation of the mechanism of JSRV oncogenesis and its hybrids have been screened by PCR for the presence or absence relationship to human bronchiolo-alveolar carcinoma. of Ͼ15,000 unique human DNA markers to generate a high resolution map of the human genome. Assuming that vector aagsiekte sheep retrovirus (JSRV) is the causative agent of a transduction correlates with the presence of the receptor gene, Jcontagious lung cancer of sheep called ovine pulmonary carci- we identified several markers that were closely linked to the noma or sheep pulmonary adenomatosis (1). Tumors originate JSRV receptor and thereby localized the receptor to the p21.3 from type II secretory alveolar and nonciliated bronchiolar epithe- band of human chromosome 3 (4). lial cells, and late stages of the disease are accompanied by the Here, we show that the JSRV receptor maps to a region of secretion of copious lung fluid containing the virus. Purified virus 3p21.3 that had previously been sequenced in a search for lung induces multifocal tumors in as little as 10 days (2), suggesting the cancer tumor suppressor genes localized to this region by analysis role of a viral oncogene. However, JSRV is a simple retrovirus with of chromosomal deletions in lung and breast cancers (6, 7), and typical gag, pol, and env genes and no known oncogenes. The viral show that the receptor is HYAL2. We have characterized the structural (Gag) and enzymatic (Pol) proteins are unlikely to be responsible, because they interact primarily with viral components, but there is precedent for alteration of cellular functions by the This paper was submitted directly (Track II) to the PNAS office. envelope (Env) protein of retroviruses, which interacts with cellular Abbreviations: JSRV, jaagsiekte sheep retrovirus; GPI, glycosylphosphatidylinositol; AP, CELL BIOLOGY components to mediate virus entry. For example, the deleted Env alkaline phosphatase; Env, envelope; PI-PLC, phosphatidylinositol-specific phospholipase C; CMV, cytomegalovirus; ␤-gal, ␤-galactosidase. protein of the Friend spleen focus-forming virus causes erythro- leukemia by binding to and activating the erythropoietin receptor See commentary on page 4285. † (3). If the JSRV Env protein was indeed oncogenic, identification S.K.R. and F.-M.D. contributed equally to this work. of the host cell receptor for JSRV would provide key insights into ¶To whom reprint requests should be addressed at: Fred Hutchinson Cancer Research Center, Room C2-105, 1100 Fairview Avenue North, Seattle, WA 98109-1024. E-mail: the oncogenic mechanism of this highly pathogenic retrovirus. [email protected]. Furthermore, the contagious nature of JSRV and its ability to The publication costs of this article were defrayed in part by page charge payment. This survive exposure to proteases and surfactants present in lung fluid article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. suggest that vectors based on JSRV might be useful for gene therapy §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.071572898 PNAS ͉ April 10, 2001 ͉ vol. 98 ͉ no. 8 ͉ 4443–4448 Downloaded by guest on October 2, 2021 receptor and show that it is a glycosylphosphatidylinositol (GPI)- 1unit͞ml, and the cells were incubated at 4°C for 4 h. Cells were anchored cell-surface protein. Although HYAL2 is related to pelleted, and the supernatants were transferred into new tubes known hyaluronidases, HYAL2 exhibited no hyaluronidase ac- containing protease inhibitor mixture (Sigma) for Western blot- tivity, and its normal function is currently unknown. Finally, we ting analysis. The pelleted cells were washed two times with PBS show that the env gene of JSRV can transform cultured fibro- and divided into two fractions for immunostaining and Western blasts, setting the stage for further work to determine the role of blot analysis. the receptor in JSRV Env-mediated oncogenesis. For Western blot analysis, the cells were lysed in 50 mM Hepes (pH 7.4), 150 mm NaCl, 10% glycerol, 1 mM EDTA, 1 mM Materials and Methods sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM Plasmid Expression Vectors. All cDNAs were expressed by using a NaF, 1% Triton X-100, 10 ␮g͞ml leupeptin, 10 units͞ml apro- cytomegalovirus (CMV) immediate early promoter and a bovine tinin, and 1 mM PMSF. Insoluble material was removed by growth hormone gene polyadenylation signal. By convention, hu- centrifugation. Flag-tagged HYAL2 was immunoprecipitated man protein names are capitalized whereas only the initial letter is from the clarified cell lysates and supernatants from PI-PLC- capitalized for those of mouse. Most were expressed by using treated cells by using anti-Flag goat antibodies (Santa Cruz pCR3.1 (Invitrogen), with the exception of mouse Hyal1, which was Biotechnology) and protein-G-agarose conjugate (GIBCO͞ expressed by using pcDNA3.1 (Invitrogen). Expression vectors for BRL). Precipitated proteins were separated by electrophoresis human HYAL4 and SPAM1 and for mouse Hyal1 and Hyal3 were in 8–16% polyacrylamide gels containing SDS and were trans- kind gifts from Antonei Csoka and Robert Stern (University of ferred to a nitrocellulose membrane; Flag-tagged HYAL2 was California, San Francisco). Accession numbers for hyaluronidase detected by using anti-Flag monoclonal antibodies (Upstate family member cDNAs are as follows: HYAL1, U03056; HYAL2, Biotechnology, Lake Placid, NY). U09577; HYAL3, AF036035; HYAL4, AF009010; human For immunofluorescence analysis, the transfected 293 cells SPAM1, L13781; Hyal1, AF011567; and Hyal2, AF302843. Amino- were placed on coverslips precoated with polylysine (molecular terminal Flag-tagged proteins were made by cloning the cDNAs weight 70,000–150,000, Sigma) and were fixed in 3.7% formal- into pFLAG-CMV2 (Flag tag without an endoplasmic reticulum dehyde in PBS for 30 min at room temperature. For some signal sequence) or pFLAG-CMV1 (preprotrypsin
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