TRANSFECTION OF THE TYPE I TGF- RECEPTOR RESTORES TGF- RESPONSIVENESS IN PANCREATIC CANCER Markus WAGNER 1,3 , Jo ¨ rg KLEEFF 1,3 , Martha E. LOPEZ 1 , Irene BOCKMAN 1 , Joan MASSAQUE ´ 2 and Murray KORC 1 * 1 Departments of Medicine, Biological Chemistry, and Pharmacology, University of California, Irvine, CA, USA 2 Cell Biology and Genetics Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Transforming growth factor-beta (TGF-) signaling is initi- ated following heterodimerization of the type II TGF- receptor (T RII) with the type I TGF- receptor (T RI). Both receptorsare required for TGF- responsiveness. In the present study, we characterized the actions of TGF-1 in T3M4 human pancreatic cancer cells, which express low levels of T RI and high levelsof T RII. Cells were transiently transfected with p3TP-Lux, a TGF--responsive luciferase reporter gene construct. T GF-1 waswithout effect in paren- tal T3M4 cells, but caused a time- and dose-dependent increase in luciferase activity in T3M4 cells co-transfected with a T RI cDNA expression vector. Co-transfection of T RI with a truncated Smad4 cDN A that is known to block TGF--dependent signaling, abrogated the T RI-induced in- crease in luciferase activity. Sequencing of the T RI and the Smad4 genes in T3M4 cells did not reveal any mutations. T hese findings indicate that one mechanism for TGF- resistance in pancreatic cancer is due to a quantitative decrease in T RI expression. Int. J. Cancer 78:255–260, 1998.  1998 Wiley-Liss, Inc. Transforming growth factor betas (TGF-s) are multifunctional polypeptides that are often characterized as negative regulators of epithelial cell growth (Massague ´, 1990; Sporn and Roberts, 1992). TGF-s also regulate cell growth and differentiation, extracellular matrix deposition, cellular adhesion properties, angiogenesis and immune functions (Massague ´, 1990; Sporn and Roberts, 1992). The TGF- gene family includes several TGF- isoforms, activins, inhibins, Mullerian inhibiting substance and bone morphogenetic proteins (Massague ´, 1990; Sporn and Roberts, 1992). Three TGF- isoforms, TGF-1, TGF-2 and TGF-3 are expressed in mamma- lian cells. These isoforms are synthesized as precursors that undergo proteolytic cleavage, resulting in the generation of biologi- cally active dimers. TGF-s exert their effects by binding to a family of transmembrane receptors that function as serine/ threonine kinases (Wrana et al., 1994). The type II TGF- receptor (TRII) is constitutively active as a serine/threonine kinase, binds TGF- in the absence of the type I TGF- receptor (TRI), but must heterodimerize with and phosphorylate TRI to initiate signaling (Wrana et al., 1994). Although there are several types of TRI, the pivotal TRI for TGF--mediated signaling is also known as ALK5 (ten Dijke et al., 1994). Activation of TRI leads to phosphorylation of Smad2 and Smad3, which then leads to heterodimerization with Smad4. The hetero-oligomeric complex is then translocated into the nucleus, where it effects transcription of specific genes either by binding directly to DNA or by complex formation with other components (Heldin et al., 1997). All 3 mammalian TGF-s are overexpressed in human pancre- atic cancers and the presence of TGF-s in the cancer cells within the tumor mass is associated with shortened patient survival post-operatively (Friess et al., 1993b). In vivo, a significant number of these cancers express high levels of TRII but low levels of TRI (Baldwin et al., 1996; Friess et al., 1993a). In vitro, cultured pancreatic cancer cell lines are often resistant to TGF-s (Baldwin and Korc, 1993), exhibit Smad4 mutations (Schutte et al., 1996) and express low levels of TRI (Baldwin et al., 1996). It has been clearly established that Smad4 mutations interfere with the signal- ing pathway that mediates TGF-1-induced growth suppression in pancreatic cancer cells (Grau et al., 1997). However, the functional significance of the quantitative decrease in TRI levels in these cells is not known. In the present study, therefore, we sequenced the TRI and Smad4 genes in T3M4 human pancreatic cancer cells that are resistant to TGF-1 and characterized their responsiveness to TGF-1 after transfecting the cells with a TRI expression vector. We now report that the TRI and Smad4 genes are not mutated in T3M4 cells and that their responsiveness to TGF-1 can be restored by increasing the level of expression of TRI. MATERIAL AND METHODS Material The following were purchased: fetal bovine serum (FBS), Dulbecco’s minimal essential medium (DMEM), RPMI medium, trypsin-ethylenediamine tetraacetic acid (EDTA) solution and penicillin-streptomycin solution from Irvine Scientific (Santa Ana, CA); oligonucleotides from Bio Synthesis (Lewisville, TX); restric- tion enzymes from Boehringer Mannheim (Indianapolis, IN); pGEM3Zf vector from Promega (Madison, WI); Sequenase kit from United States Biochemical (Cleveland, OH); - 32 P-CTP (400 Ci/mmol), - 32 P-dCTP (3,000 Ci/mmol), - 35 S-dATP (1,000 Ci/ mmol), ThermoSequenase radiolabeled terminator cycle sequenc- ing kit, and ECL blotting kit from Amersham (Arlington Heights, IL); polyclonal rabbit anti TRI, and anti TRII antibodies (V22: anti TRI; H567: anti TRII) from Santa Cruz Biotechnology (Santa Cruz, CA); Immobilon PVDF transfer membranes from Millipore (Bedford, MA); a random prime labeling kit from Boehringer-Mannheim (Indianapolis, IN); GeneScreen membranes from New England Nuclear (Boston, MA); pBluescript-IISK + from Stratagene (La Jolla, CA); beetle luciferin from Promega; and reverse transcription assay kit and lipofectamine from GIBCO- BRL (Gaithersburg, MD). T3M4 and COLO-357 human pancreatic cancer cells were a gift of Dr. R.S. Metzger (Duke University, Durham, NC). Mv1Lu mink lung epithelial cells were from Dr. D. Rifkin (New York University School of Medicine, New York, NY). Recombinant human TGF-1 was a gift from Genentech (South San Francisco, CA). All other chemicals and reagents were of tissue culture grade and purchased from Sigma (St. Louis, MO). Cell culture and proliferation assay T3M4 cells were maintained in RPMI medium. COLO-357 and Mv1Lu mink lung epithelial cells were maintained in DMEM medium. Media contained 10% FBS, penicillin G (100 U/ml) and streptomycin (100 μg/ml). Cells were seeded overnight in 96-well plates at 10,000 cells/well, and then incubated for 24, 48 or 72 hr with medium containing 0.1% BSA, 5 μg/ml transferrin, 5 ng/ml sodium selenite and antibiotics (serum-free medium) in the absence 3 The first two authors contributed equally to this work. Grant sponsor: National Cancer Institute; Grant number: USPHS Grant CA-75059. *Correspondence to: Division of Endocrinology, Diabetes and Metabo- lism, Medical Sciences I, C240, University of California, Irvine, CA 92697, USA. Fax: (949) 824–1035. Received 19 March 1998; Revised 11 May 1998 Int. J. Cancer: 78, 255–260 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer