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Angiogenesis, Metastasis, and the Cellular Microenvironment

Induction of Tumorigenesis and Metastasis by the Murine Orthologue of Tumor Protein D52

¹ Department of Microbiology and Immunology and the Southwest Cancer Treatment and Research Center, Texas Tech University Health Sciences Center, Lubbock, Texas; ² Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Portland, Oregon; ³ The University of Sydney Discipline of Paediatrics and Child Health, Childrens Hospital at Westmead, NSW, Australia; ⁴ Department of Laboratory Medicine and Pathology, Mayo Foundation and Clinic, Rochester, Minnesota; and ⁵ Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Minnesota, Minneapolis-St. Paul, Minnesota

Requests for reprints: Robert K. Bright, Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, 3601 4th Street, MS 6591, Lubbock, TX 79430. Phone: 806-743-4592; Fax: 806-743-2334; E-mail: robert.bright{at}ttuhsc.edu

Expression studies have consistently identified tumor protein D52 (TPD52) overexpression in tumor cells. Murine TPD52 (mD52) shares 86% identity with the human orthologue. To study a possible role for TPD52 in transformation, 3T3 fibroblasts were transfected with the full-length cDNA for mD52. Expression of mD52 was confirmed by reverse transcription-PCR (RT-PCR), real-time PCR, and Western blot analysis compared with 3T3 and vector-transfected 3T3 (3T3.V), and the resultant cell line was designated 3T3.mD52. At 4 weeks, 3T3.mD52 gained a 2-fold increase in growth rate, lost contact inhibition, and exhibited a marked phenotype change. Further characterization revealed an acquired ability for anchorage-independent cell growth. To determine whether 3T3.mD52 had become tumorigenic, naïve, healthy, immunocompetent syngeneic mice were inoculated subcutaneously with varying cell doses. Tumors measuring >1 cm² were detected 60 days postinoculation with 3T3.mD52, and a 50% subcutaneous tumor incidence was obtained with as few as 5 x 10⁵ 3T3.mD52 cells. Remarkably, when lungs from 3T3.mD52 tumor-bearing mice were analyzed, numerous tumor nodules were observed, ranging from nodules less than 10 to nodules too numerous to count (inoculation with 1 x 10⁵ and 5 x 10⁶ cells, respectively). Further support for the metastatic capacity of 3T3.mD52 was the demonstration that transforming growth factor (TGF)-ßR1 (receptor) expression decreased and TGF-ß1 secretion increased in 3T3.mD52 compared with 3T3 controls. cDNA microarray analysis showed a gene expression pattern that further supported mD52-induced transformation and metastasis. Together, these data suggest that mD52 expression in 3T3 cells initiated cellular transformation, tumorigenesis, and progression to metastasis. (Mol Cancer Res 2007;5(2):133–44)