Int. J. Cancer: zyxwvutsrqpo 67,283-288 (1996) zyxwvutsr 0 1996 Wiley-Liss, Inc. zyxwvutsrq Publication zyxwv d zyxw Ihe zyxwv lnlernabonal Union Against Cancer 1 Publication de runion Internationale antre 18 Cancer ATTENUATED ALKS RECEPTOR EXPRESSION IN HUMAN PANCREATIC CANCER: CORRELATION WITH RESISTANCE TO GROWTH INHIBITION Rae Lynn BALDWINI-3, Helmut FRIESS~, Munehiro YOKOYAMA~, Martha E. LOPEZ’, Michael S. KOBRIN’, Markus W. BUCHLER~ and Murray KORC1v4 ‘Division of Endocrinology,Diabetes and Metabolism, Departments of Medicine and Biological Chemistry, Universiv of California, Irvine, zyxwvutsrqp CA 9271 7; and 2Department of Viiceral and TransplantationSurgery, Universiv of Berne, CH-3010 Beme, Switzerland Transforming growth factor-6 (TGF-p) receptors constitute a family of transmembrane proteins that bind TGF-P ligands. In this study we assessed the growth responsiveness to TGF-P I in pancreatic cancer cell lines and characterized the levels of expression of TGF-f3 receptors in these cell lines and in human pancreatic cancer tissues. COLO 357 cells were most sensitive to the growth inhibitory actions of TGF-PI, PANC-I cells exhibited moderate semitii, Hs766T cells exhibited slight sensitivity and MIA PaCa-2 and T3M4 cells were resistant to TGF-PI. Only COLO 357 cells expmsed high levels of ALK5, the major type I TGF-P receptor (TPRI). Hs766T and PANC- I cells expressed high levels of SKRl, another TPRl subtype. Only MIA PaCa-2 cells did not exhibit the type II TGF-P receptor (TP-RII) transcript, whereas type 111 TGF-f3 receptor (TP-RIII) mRNA levels were elevated in this cell line and in HS7661cells. All the cell lines expressed TGF-PI, but TGF-PI and TGF-fl3 mRNA levels were variable. ALK5 and SKR I mRNA levels were 6.8- and 9-fold greater in the pancreatic tumors in comparison with the corresponding levels in the normal pancreas. However, in the cancer cells, ALK5 immunoreactivity was faint, whereas TPRll immunoreactivity was focal and intense. Conversely, in ductal cells adjacent to cancer cells ALK5 immunoreactivitywas strong, whereas TPRll immunoreactivky was weak. Since ALK5 heterodimerization with TPRll is crucial for TGF-fbmediated signaling, our findings suggest that low levels of ALK5 in pancreatic cancer cells within a tumor may protect against growth inhibition. Q 1996 Wiley-Liss, Inc. Transforming growth factors-beta (TGF-Bs) are multifunc- tional polypeptides that are often characterized as negative regulators of epithelial cell growth (Sporn and Roberts, 1992). TGF-ps also regulate cell growth and differentiation, extracel- Mar matrix deposition, cellular adhesion properties, angiogen- esis and immune functions (Sporn and Roberts, 1992). The TGF-P supergene family includes several TGF-P isoforms, activins, inhibins, Mullerian inhibiting substances and bone morphogenetic proleins (Sporn and Roberts, 1992). Three TGF-P isoforms, TGF-PI, TGF-P2 and TGF-P3, are ex- pressed in mammalian cells. They are synthesized as precur- sors that undergo proteolytic cleavage, resulting in the genera- tion of biologically active dimers (Sporn and Roberts, 1992). The mature forms of TGF-P1 and 432 share 70% amino acid sequence homology. and the mature form of TGF-P3 shares 80% homology with the other two TGF-Ps (Sporn and Rob- erts, 1992). TGF-ps exert their effects by binding to a family of transmem- brane receptors that function as serine/threonine kinases (Lin et al., 1992; Franzen et al., 1993). The type I1 TGF-P receptor (TP-RII) binds TGF-P in the absence of the type I TGF-P receptor (TP-RI), but its kinase activity requires the presence of TP-RI (Wrana et d., 1994). While only one TP-RII has been identified, several TP-RI receptors have been cloned and characterized (Attisano et al., 1993). Their extracellular do- main is shorter than that of TP-RII, and, in contrast to TP-RII, they possess a highly conserved GS motif in their intracellular juxtamembrane region. The pivotal TP-RI receptor for TGF-P- mediated signaling is ALKS (ten Dijke et al., 1994). Another TP-RI receptor subtype, SKRl (Matsuzaki et al., 1993), can also mediate TGF-P1 effects, but to a lesser extent than ALK5 (Ebner et al., 1993). There are also several TGF-P binding proteins that do not possess signaling activity, including the type 111 TGF-f3 receptor (TP-RIII), which is also known as betaglycan (Lopez-Casillas et al., 1993). In contrast to TP-RI and TP-RII, which bind TGF-Ps in their extracellular cysteine- rich domains, TP-RIII is a membrane proteoglycan that binds TGF-f3 through its core protein and functions to present TGF-Ps to TP-RI and TP-RII. Pancreatic ductal adenocarcinoma is a devastating disease associated with poor prognosis. All three TGF-B isoforms are overexpressed in human pancreatic tumors, where their pres- ence is associated with shortened patient survival postopera- tively (Friess et al., 19936). In addition, TPR-I1 mRNA levels are elevated in pancreatic cancers (Friess et al., 1993~). Conceivably, TGF-ps act to suppress cancer-directed immune mechanisms while promoting angiogenesis and modulating the extracellular matrix in a manner that enhances cancer spread. It is not known, however, how pancreatic cancer cells avoid TGF-P-mediated autocrine and paracrine suppression of their growth. Because TGF-P action is dependent on the presence of TGF-P receptors, in the present study we characterized the expression of these receptors in human pancreatic cancer cell lines and tissues. We now report that responsiveness to TGF-B1-mediated growth inhibition in cultured human pancre- atic cancer cell lines correlates with the presence of high levels of ALK5, and that malignant cells within human pancreatic cancers express high levels of TP-RII but low levels of zy ALKS. MATERIAL AND METHODS Material Fetal bovine serum (FBS) was from Gemini BioProducts (Calabasas, CA). Dulbecco’s minimal essential medium (DMEM), RPMI medium, trypsin EDTA solution and penicil- lin-streptomycin solution were from Irvine Scientific (Santa h a , CA). Oligonucleotide synthesis was performed with a model 391 DNA synthesizer (Applied Biosystems, Foster City, CA) with reagents purchased from Applied Biosystems. Restric- tion enzymes were from Boehringer Mannheim (Indianapolis, IN), pGEM3Zf vector was from Promega (Madison, WI) and sequencing was done with a Sequenase kit from USB (Cleve- land, OH). A random prime labeling kit was from Boehringer- Mannheim. Genescreen hybridization membranes were from NEN (Boston, MA). Anti-ALKS and anti-TP-RII antibodies were from Santa Cruz (Santa Cruz, CA). The streptavidin peroxidase kit was from Kirkegaard and Perry (Gaithersburg, MD). Hs766T, MIA PaCa-2 and PANC-1 human pancreatic cells were obtained from the ATCC (Rockville, MD). COLO Current address: Department of Obstetrics and Gynecology, Cedars-SinaiMedical Center, 8700 Beverly Boulevard, Suite 1738, Los Angeles, CA 90048. 4T0 whom correspondence and reprint requests should be ad- dressed, at the Division of Endocrinology,Diabetes and Metabolism, Medical Sciences I, C240, University of California, Irvine, CA 92717. Fax: (714) 824-2200. Received: January 18,1996 and in revised form March 5,1996.