EXPRESSION OF SMAD PROTEINS IN HUMAN COLORECTAL CANCER Olexander KORCHYNSKYI 1,2 , Mare ´ne LANDSTRO ¨ M 1 , Rostyslav STOIKA 2 , Keiko FUNA 3 , Carl-Henrik HELDIN 1 , Peter TEN DIJKE 1 and Serhiy SOUCHELNYTSKYI 1 * 1 Ludwig Institute for Cancer Research, Uppsala, Sweden 2 Institute of Biochemistry of the National Academy of Sciences of Ukraine, Lviv, Ukraine 3 Institute of Anatomy and Cell Biology, Go ¨teborg University, Go ¨teborg, Sweden Escape from transforming growth factor- (TGF-)– induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF- family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohisto- chemistry in human colorectal cancers. W e found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Com- mon-mediator Smad4 and inhibitory Smadswere detected in cells of both tumor and normal tissues. W e observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal can- cers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts. Int. J. Cancer 82:197–202, 1999.  1999 Wiley-Liss, Inc. Transforming growth factor- (TGF-) family members are involved in the regulation of cell proliferation, differentiation, motility and apoptosis (Roberts and Sporn, 1990). TGF- is a potent inhibitor of cell growth, and resistance to TGF--induced growth inhibition is often observed in tumor cells (Roberts and Sporn, 1990; Lahm and Odartchenko, 1993). Reduced responsive- ness to TGF- was reported to be an important event in colorectal carcinogenesis (Manning et al., 1991). Loss of cell sensitivity to TGF- was found to be associated with mutations of specific receptors or deregulation of intracellular signaling pathways (for review: Roberts and Sporn, 1990; Lahm and Odartchenko, 1993; Derynck and Feng, 1997; Heldin et al., 1997; Massague ´, 1998). Smad proteins play a pivotal role in intracellular TGF- signaling (for review: Heldin et al., 1997; Massague ´, 1998). Eight mammalian Smads have been described and divided into 3 groups based on their functions in signal transduction. The first group consists of receptor-activated Smads, which are specifically phos- phorylated by different type I serine/threonine receptors of the TGF- family. Smad2 and Smad3 are phosphorylated by activated type I receptors for TGF- and activin; Smad1, Smad5 and Smad8 are phosphorylated by activated type I receptors for bone morpho- genetic proteins (BMPs). Another type of Smad molecule, a common-mediator Smad4, forms complexes with receptor- activated Smads. These complexes are translocated to the cell nuclei, where they regulate expression of target genes. The third Smad category consists of Smad6 and Smad7, which play inhibi- tory roles in TGF- signaling by blocking phosphorylation of receptor-activated Smads or by competitive inhibition of complex formation of receptor-activated Smads with the common-mediator Smad4 (for review: Derynck and Feng, 1997; Heldin et al., 1997; Massague ´, 1998). Inactivating mutations of Smad proteins have been found in human cancers. In particular, Smad4 is altered in pancreatic and colorectal cancers. Alterations of receptor-activated Smad2 and Smad3 in tumor cells have also been described (for review: Derynck and Feng, 1997; Heldin et al., 1997; Massague ´, 1998). Mutations of Smad proteins render them inactive, thus providing resistance of cells to TGF--induced growth inhibition. To date, no systematic investigation of the expression of Smad proteins of all groups in the same specimen of human cancer has been reported. We addressed this question by studying expression of all Smads in specimens of human colorectal cancer by immunohistochemistry. Up-regulation of receptor-activated Smad proteins in a fraction of the tumor cells was found, while common-mediator Smad4 and inhibitory Smad6 and Smad7 were detected in tumor cells as well as in cells of normal colon. We found high expression of common-mediator Smad4 in columnar absorptive cells of the surface epithelium but not in the zones of active cell proliferation. This suggests a role for Smad4 in terminal differentiation and apoptosis of epithelial cells of colon crypts. MATERIAL AND METHODS Tissue specimens Specimens of human colorectal tumor tissues and adjacent morphologically normal tissues were collected at the Department of Proctology, Lviv State Medical University, Ukraine. Adjacent morphologically normal tissues were collected 10 to 15 cm from tumor tissues and did not manifest any histological characteristics of transitional mucosa (Filipe, 1984). Immediately after resection, tissues were fixed overnight in 4% formaldehyde and paraffin- embedded according to standard techniques (Hogan et al., 1994). Sections of 4 μm were cut and collected on silane-coated glass slides. The grade of malignancy was histopathologically classified according to the TNM classification (UICC, 1987). Among the 14 specimens collected, 11 were T 3 , one was T 2 and one was T 4 grade. In no case were distal metastases detected. Antibodies Rabbit anti-sera raised against peptides derived from linker regions of Smad1 (BSP/TFP) and Smad5 (SSN) have been described by Tamaki et al. (1998). Anti-sera raised against peptides derived from linker regions of Smad2 (DQQ), Smad3 (DHQ) and Smad4 (HPP) have been described by Nakao et al. (1997). Anti-serum against Smad7 (KER) was raised against the synthetic peptide KERQLELLLQAVESRGGTRTA. Each anti-serum to re- ceptor-activated and common-mediator Smad4 has been found to be specific to respective Smad proteins and to not cross-react with other Smads, except for the Smad1 anti-serum, which cross-reacts with Smad8 (Smad1/8 anti-serum), and the Smad7 anti-serum, which cross-reacts with Smad6 (Smad6/7 anti-serum), by Western blotting assays. Anti-sera against Smads were affinity-purified using CNBr-activated Sepharose CL-4B (Pharmacia, Uppsala, Sweden) with immobilized specific peptides according to the manufacturer’s protocol. Affinity-purified rabbit polyclonal anti- Grant sponsor: Royal Swedish Academy of Sciences; Grant number: 1390. *Correspondence to: Ludwig Institute for Cancer Research, Box 595, 751 24, Uppsala, Sweden. Fax.: +46 18 16 04 20. E-mail: sergiy@licr.uu.se Received 8 December 1998; Revised 5 February 1999 Int. J. Cancer: 82, 197–202 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer