(CANCER RESEARCH SS. 4592-4597. October 15. 19981 Advances in Brief Dpc4 Transcriptional Activation and Dysfunction in Cancer Cells1 Jia Le Dai, Kenan K. Turnacioglu, Mieke Schutte, Avrahom Y. Sugar, and Scott E. Kern2 De/Hirtments ¡if Oncology ¡M.S., S. E. K.I and Pathology ¡J.L D.. K. K. T.. A. Y. S., S. E. K.], The Johns Hopkins Medical Institutions. Baltimore. Maryland 2I205-2I96 Abstract Dpc4 (Smad4) is implicated in mediation of signals from transforming growth factor (TGF) /! and related ligands, and wild-type Dpc4 mediates TGF-ß-stimulated gene transcription at specific DNA sequences bound by Dpc4 [Smad binding element (SBE)|. We characterized panels of I>!'(-! tumor mutations and cancer cell lines. Amino acid substitutions within the NH2-terminal third of Dpc4 weakened or ablated SBE-mediated gene regulation by a disruption of DNA binding. An interaction of the COOH- terminal end with the DNA-binding domain of Dpc4 was evident but was not required to explain the functional impairment produced by ML- terminal DPC4 mutations. Both substitution and truncation mutations of the ( '(>()! I-terminal half of DPC4 lacked the ability to regulate transcrip tion while retaining the sequence-specific DNA-binding function, but through differing mechanisms. A modular domain to redistribute Dpc4 to the nuclear compartment allowed SBE-mediated transcriptional activa tion in a cell line having a TGF-ß receptor defect and was sufficient to restore SBE-mediated transactivation ability to COOH-terminal DPC4 missense mutants. Cells harboring /»/'(/ alterations had a universal impairment of the H.I -/¡-slmmbu-d SBE transcriptional response. These studies identify the loss of SBE-mediated gene regulation as a uniform outcome of the selection for !>!'( -t alterations during tumorigenesis. They raise the possibility of restoration of some Dpc4-associated transcriptional events in cancer cells through the targeted redistribution of wild-type and some missense mutant forms of Dpc4 to the nucleus. Introduction Defects in the TGF'-ß pathway are highly prevalent in tumors and occur at multiple levels, involving mutations and aberrant expression of TGF-/3 receptors and genetic alterations of Smad genes (1-5). The DPC4 gene (SMAD4, MADH4) was cloned as a target of homozygous deletion in pancreatic, colorectal, and occasional other cancers (2). A number of lines of evidence suggest Dpc4 activity as a major down stream determinant of the TGF-ßtumor suppressor function. Dpc4 protein is structurally similar to a family of related (Smad) proteins conserved among diverse species (5-9). These similarities localize to an NH2-terminal region termed MH1 (Mad homology 1) and a COOH-terminal half, MH2 (Fig. 1). The MH1 domain of Smad3 and Dpc4 harbor sequence-specific DNA-binding properties (10, 11). The MH2 mediates interactions among Smad proteins (12, 13), harbors a transcriptional activation domain ( 14), and mediates nuclear localiza tion (15). Smad proteins can mediate or mimic signaling that is initiated by ligands of the TGF-ßsuperfamily, including bone mor- phogenetic proteins, activin, and TGF-ß.Pancreatic and colorectal cancers share the TGF-ßunresponsiveness of many other cancer types (1, 16, 17). Received 7/13/98; accepted 8/31/98. 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. ' This work was supported by NIH Grant CA68228. - To whom requests for reprints should be addressed, at 632 Ross Building. The Johns Hopkins University School of Medicine, Baltimore, MD 2I205-2196. Phone: (410) 614- 3314; Fax: (410)614-0671; E-mail: sk@welchlink.welch.jhu.edu. 1The abbreviations used are: TGF. transforming growth factor; 4-OHT. 4-hydroxyta- moxifen; SBE, Smad-binding element; MHI and MH2. Mad-homology domains 1 and 2, respectively; RT-PCR, reverse transcription-PCR. A number of properties of the Dpc4 protein have been identified. Dpc4 binds Smads 1-3, cooperating in signal transduction events that originate from various ligands (12, 13). Nuclear localization of Dpc4 is under regulatory control ( 15, 18). More direct evidence for a role in nuclear events is provided by the ability of the COOH terminus of Dpc4 to activate transcription when fused to a modular DNA-binding domain (14). None of these properties are uniformly deficient among cells harboring natural DPC4 alterations, and instead a constellation of distinguishable functional defects is apparent. Recently, Dpc4 was found to bind to sequences within the TGF-ß-responsive construct p3TP-lux (19), and with greater avidity and specificity, to a palin- dromic sequence termed the SBE that can confer Dpc4-dependent transcriptional activation to a minimal promoter (10). The delineation of this latter activity raised the possibility of a universal functional target for DPC4 mutational events and upstream TGF-ßsignaling defects. A physiological significance for the SBE is already apparent because Dpc4 binds to related repeated sequences in the TGF-ß- inducible elements in the human plasminogen activator inhibitor-type I gene promoter to activate its transcription (11, 19). During human tumorigenesis, point mutations cause premature truncation and amino acid substitutions of the COOH-terminal MH2 region and, at lesser frequency, substitutions in the NH-,-terminal MH1 region of Dpc4 in colorectal and pancreatic tumors (Refs. 2-4, 20, and 21; Fig. 1). Presumably, each of these mutations must inac tivate a function required for tumor suppression, resulting in a general loss of TGF-ßgrowth suppression that characterizes some DPC4-mi\\ pancreatic cancer cell lines (17). A comprehensive study of the functional impairments produced by these mutations has not been performed, and defects in SBE binding have not been evaluated. Artificial deletions of the COOH terminus are reported to affect nuclear localization and Smad interactions (15, 22), but the implica tions of the MH2 domain missense mutations found in tumors have not been explored. Because the missense mutations of tumors repre sent a more stringent means to test for lost tumor-suppressive func tions than the analysis of deletions (which bluntly affect many func tions), a comprehensive analysis that includes missense mutations represents a crucial test of the functional significance of the SBE transactivation ability in tumor biology. Furthermore, the low rate of DPC4 missense mutations in many tumor types requires a functional assay to address whether some alterations might represent only chance variants that do not affect protein function. A mechanistic understand ing of the natural defects in Dpc4 function found in tumors might, in turn, indicate various means to restore the downstream transcriptional output of the TGF-ßtumor-suppressive signal. Materials and Methods DNA Constructs. The DPC4 cDNA from a human library (2) was modi fied at three nucleotides within a PCR primer site to match a consensus sequence (Kozak sequence, ccaccATGG) for the start site of translation. The 1.6-kb open reading frame was subcloned into the B«mHIand EcoRl sites of pcDNA3.1 (Invitrogen), resulting in pDPC4-WT. Tumor-derived mutation sequences were introduced within the DPC4 coding region of pDPC4-WT by site-directed mutagenesis (Quick-change; Stratagene). SMAD2 and SMADJ cDNAs were similarly modified with the consensus Kozak sequence and 4592 Research. on December 6, 2021. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from