[CANCER RESEARCH 60, 1552–1556, March 15, 2000] Advances in Brief Transfection of Constitutively Active Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase Confers Tumorigenic and Metastatic Potentials to NIH3T3 Cells 1 Danny R. Welch, 2 Toshiyuki Sakamaki, Ralph Pioquinto, Timothy O. Leonard, Steven F. Goldberg, Qiufang Hon, Raymond L. Erikson, Manuel Rieber, Mary Strasberg Rieber, Deana J. Hicks, Joseph V. Bonventre, and Alessandro Alessandrini 2 Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-2390 [D. R. W., T. S., T. O. L., S. F. G., D. J. H.]; Medical Services, Massachusetts General Hospital, Charlestown, Massachusetts [R. P., J. V. B., A. A.]; Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts [Q. H., R. L. E.]; and Instituto Venezolano Investigaciones Cientificas, Caracas, Venezuela [M. R., M. S. R.] Abstract Cellular growth and differentiation are controlled by multiple extra- cellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK ki- nase). MEK1 is activated by phosphorylation at Ser 218 and Ser 222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp 218 ], [Asp 218 , Asp 222 ], and [Glu 218 , Glu 222 ], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The trans- formed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp 218 ] or [Asp 218 , Asp 222 ] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metasta- ses. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mecha- nism through which ras could confer tumorigenic and metastatic potential (i.e., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1). Introduction Components of the MAP 3 kinase signaling pathways (e.g., gip2, Ras, and Raf) cause oncogenic transformation in their constitutively active forms (1). Moreover, expression of activated ras can confer metastatic potential upon some cells (reviewed in Ref. 2). The pur- poses of this study were: (a) to begin to ascertain what the down- stream effectors of ras transformation and ras-induced metastatic potential are; and (b) to use stable Mek1 mutant variants to address whether ERK1/2 activity is essential for these phenotypes. Expression of constitutively active MEK1 in NIH3T3 fibroblasts results in cellular transformation (3). Activation of MEK1 is accom- plished by phosphorylation of serines at positions 218 (S218) and 222 (S222; Ref. 3). To create constitutively active MEK1, S218 and S222 were mutated to aspartic acid, mimicking the phosphorylated/active state. The MEK1-activated mutants were designated DS and DD, where DS is Asp218/Ser222 and DD is Asp218/Asp222. DS and DD clonal cell lines produced colonies when grown in soft agar, an in vitro indicator of transformation. However, anchorage-independent growth did not correlate with ERK1/2 activity. The DS (DS2 and DS4) lines exhibited constitutively active ERK1/2, yet yielded fewer colonies compared with DD lines (DD1 and DD3), which had basal ERK1/2 activity. These data suggested that maintenance of transfor- mation was independent of ERK1/2 activity. Recently, Webb et al. (4), using various ras mutants, showed that although tumorigenicity was independent of ERK1/2 activity, metas- tasis required its activation. Therefore, we wanted to determine whether clonal cell lines that we established previously and that exhibited constitutive or basal levels of ERK1/2 activity could also confer tumorigenicity and/or metastatic potential. Our data show that tumorigenic and metastatic potentials are dependent upon MEK1 activation but appear to be independent of ERK1/2 activity. Materials and Methods Analysis of Metastatic Potential of MEK1-transformed Clonal Lines. Single-cell suspensions of DS and DD were made in ice-cold HBSS and were injected into the lateral tail veins of female athymic mice, 3– 4 weeks of age, in a total volume of 0.2 ml/mouse. Mice were killed by methoxyflurane (Metofane; Pitman-Moore, Washington Crossing, NJ), followed by cervical dislocation. Complete necropsies were performed, and metastases were quan- tified as described (5). Lung metastases were counted after fixing whole lungs in a mixture of neutral buffered formalin and Bouin’s fixative (5:1). Random 4–6-m H&E-stained sections were examined. All animal studies were per- formed according to the guidelines of the NIH, and protocols were approved by the Institutional Animal Care and Use Committee. Western Blot. Lungs were isolated 27– 41 days after i.v. inoculation from NIH3T3-, DS- and DD-injected athymic mice. The tissue was analyzed by Western blotting as described (3) with some modifications. Briefly, lungs were lysed by Dounce homogenization in potassium phosphate buffer [10 mM KPO 4 (pH 7.05), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl 2 , 50 mM -glycerophos- phate, 1 mM sodium vanadate, 1 mM DTT, 0.5% NP40, 0.1% Brij-3, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml pepstatin A]. Lung metastases were dissected from adjacent normal tissues before further processing. Lysates were centrifuged at 16,000  g for 10 min. Supernatants (40 g) were boiled in 1 sample buffer [500 mM Tris-HCl (pH 6.8), 10% SDS, 20% glycerol, 0.05% bromphenol blue, and 1% 2-mercapto- Received 12/13/99; accepted 2/2/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants CA62168 (to D. R. W.), NS10828 (to A. A. and J. V. B.), DK39773 (to J. V. B.) from the NIH; the United States Army Medical Research and Materiel Command DAMD17-96-1-6152 (to D. R. W.); the National Foundation for Cancer Research (to D. R. W.); The American Heart Association—Massachusetts Divi- sion; and CONICIT grants S1-96001340 and G-9700613 (to M. R. and M. S. R.). 2 To whom requests for reprints should be addressed, D. R. W. at Jake Gittlen Cancer Research Institute, Room C7810, Box H-059, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033-2390 or A. A. at Renal Unit, Massachusetts General Hospital-East, 149 13th Street, Charlestown, MA 02129. 3 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; DS, MEK1-DS; DD, MEK1- DD; MMP, matrix metalloproteinase. 1552 Research. on February 29, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from