ANTICANCER RESEARCH 28 : 1531-1540 (2008) Review TGFβ-regulated Gene Expression by Smads and Sp1/KLF-like Transcription Factors in Cancer VOLKER ELLENRIEDER Signal Transduction Laboratory, Internal Medicine, Department of Gastroenterology and Endocrinology, University of Marburg, Marburg, Germany Abstract. Transforming growth factor beta (TGF β) controls complex induces the canonical Smad signaling molecules which vital cellular functions through its ability to regulate gene then translocate into the nucleus to regulate transcription (2). The expression. TGFβ binding to its transmembrane receptor cellular response to TGF β can be extremely variable depending kinases initiates distinct intracellular signalling cascades on the cell type and the activation status of a cell at a given time. including the Smad signalling and transcription factors and also For instance, TGF β induces growth arrest and apoptosis in Smad-independent pathways. In normal epithelial cells, TGF β healthy epithelial cells, whereas it can also promote tumor stimulation induces a cytostatic program which includes the progression through stimulation of cell proliferation and the transcriptional repression of the c-Myc oncogene and the later induction of an epithelial-to-mesenchymal transition of tumor induction of the cell cycle inhibitors p15 INK4b and p21 Cip1 . cells (1, 3). In the last decade it has become clear that both the During carcinogenesis, however, many tumor cells lose their tumor suppressing and the tumor promoting functions of TGF β ability to respond to TGF β with growth inhibition, and instead, are primarily regulated on the level of gene expression through activate genes involved in cell proliferation, invasion and Smad-dependent and -independent mechanisms (1, 2, 4). In metastasis. Strong efforts have been made during recent years to many cases, receptor activation not only stimulates the characterize Smad-mediated transcriptional processes and to downstream Smad cascade, but also Smad independently identify those TGFβ-regulated transcription factors that control signalling and transcription molecules which might work gene expression independent of the Smads. These studies have together or independent from each other to regulate gene led to the identification of a novel family of TGFβ-inducible expression in response to TGF β (3, 4). In addition, TGF β Sp1/KLF-(Krüppel-like factors) like transcription factors induces the expression of early response transcription factors (KLF10 and KLF11) which play remarkable roles in TGFβ such as the Sp1/KLF-(Krüppel-like-factors) like zinc-finger mediated cell growth control and differentiation. In this article, proteins KLF10 and KLF11 which work as effector proteins in the current knowledge on the peculiar roles of Sp1/KLF-like TGF β mediated cell growth control and differentiation (5, 6). proteins in Smad dependent and -independent gene regulation Following induction by TGF β, KLF11 represses the expression initiated by TGFβ, are summarized. of the c-Myc oncogene, terminates the negative Smad7 feedback loop and renders cells more sensitive to oxidative drugs through Transforming growth factor- β (TGF β) is a pleiotropic cytokine silencing of the oxidative scavengers superoxide dismutase 2 that regulates a wide variety of cellular processes including cell (SOD2) and Catalase1 (7, 8). Taken together, the recent studies growth and apoptosis, differentiation, migration, and metastasis suggest a model in which the rapid induction of KLF11 (1). TGF β binding to its transmembrane receptor kinase contributes to TGF β-induced cell growth inhibition through different transcriptional mechanisms including concerted actions with the Smads but also Smad-independent mechanisms. Correspondence to: Volker Ellenrieder, MD, Department of Internal TGF β – Activation of Effector Signalling and Medicine, Gastroenterology and Endocrinology, University of Transcription Pathways Marburg Baldinger Strasse 35043 Marburg, Germany. Tel: +49 0 64212866460, Fax: +49 0 64212868922, e-mail: [email protected] marburg.de The prototype of the TGF β family, TGF β1, binds with high affinity to the type-II TGFβ receptor (TβR-II), which then Key Words: TGF β, Smads, Sp1/KLF-like transcription factors, gene recruits the type-I TGFβ receptor (TβR-I) to form a expression, review. heteromeric complex (1, 9). This three-component module 0250-7005/2008 $2.00+.40 1531 ANTICANCER RESEARCH 28 : 1531-1540 (2008) then allows the kinase activity of the type-II receptor to stimulation, Smads constantly undergo cycles of receptor- phosphorylate the TGFβ receptor and subsequently to bind mediated phosphorylation (activation) and phosphatase- signalling molecules such as members of the Smad family of mediated dephosphorylation, resulting in sustained nuclear- transcription factors (9). Although there are considerably cytoplasmic shuttling of the transcription factors (15). fewer receptors and Smads than there are ligands, a greater Receptor-mediated Smad activation and nuclear translocation versatility of signalling is possible than might be expected. is finally terminated by inhibitory Smads, Smad6 and Combinatorial interactions of type I and type II receptors and Smad7, which serve as negative feedback loops in TGFβ Smads in oligomeric complexes allow substantial diversity, signalling (16). Upon induction by TGFβ or other growth and are complemented by the many sequence-specific factor signals, Smad7 binds to the activated type-I TGFβ transcription factors with which Smads cooperate, resulting receptor in competition with R-Smads and thereby blocks in context-dependent transcriptional regulation (2, 10). In further activation and downstream signal transduction of the addition to the Smad signaling pathway, activation of the Smads (16). receptor complex initiates signalling through Smad Genetic alterations of this simple TGF β signalling independent mechanisms (4). These alternative, non-Smad pathway are frequently found in somatic and heritable pathways can either work in concert with or independent disorders as well as in various tumor entities. Inactivating from Smad proteins to regulate vital cellular functions mutations of the type-II receptor, for instance, have been through transcriptional responses to TGFβ (4, 7, 10). Finally, reported in colorectal, gastric and endometrial carcinomas TGFβ can induce immediate early response transcription and in association with a more aggressive phenotype (17). factors, such as the KLF10 and KLF11 zinc-finger proteins, In addition, more than half of pancreatic carcinomas and which function as nuclear regulators to repress or activate approximately one third of colorectal carcinomas harbor gene expression together with or irrespective of the Smad inactivating mutations of the Smad4 gene based on proteins (5, 6). homozygous deletion or intragenic mutation (18-20). In many cases, genetic loss of Smad4 causes disrupted The Smad Signalling Pathway in Normal nuclear-cytoplasmic shuttling of Smad complexes and thus and Malignant Epithelial Cells renders tumor cells insensitive to nearly all TGFβ-regulated transcriptional responses, including those associated with Smad proteins are central downstream effectors of TGF β growth inhibition. Genetic alterations of the Smad4 gene signaling in normal and transformed cells. Smads exist as are also found in familial juvenile polyposis, an autosomal three subgroups: Receptor-Smads ( e.g. Smad2 and Smad3), dominant disease characterized by predisposition to common Smads ( e.g. , Smad4), and inhibitory Smads (Smad6 gastrointestinal polyps and cancer (21). In this syndrome, and Smad7) (9). Smad2 and Smad3 serve as receptor- polyps are formed by inactivation of the Smad4 gene activated signalling molecules which transduce the TGFβ- through germline mutation and loss of the unaffected wild- stimulus into the nucleus following interaction with Smad4. type allele. Together, disruption of TGFβ-Smad signalling Inactive R-Smads are anchored to the plasma membrane through inactivating mutations is commonly observed in through various molecules, among which the Smad anchor different malignancies and is closely associated with the for receptor activation (SARA) has been most extensively loss of tumor suppression by TGFβ (22). Importantly, studied (11). Upon ligand-binding, the activated TGFβ type- however, signal transduction and target gene transcription I (T βR-I) kinase phosphorylates the C-terminal Ser-Ser-X- by the Smads is not only affected by genetic alterations Ser motif of R-Smads, allowing them to form heteromeric within the pathway, but also through signalling crosstalk complexes with Smad4 and to move into the nucleus in order interactions. In fact, at one level or another, essentially all to regulate the expression of selected TGFβ target genes (9, major tumor suppressor and oncogenic signaling pathways 11, 12). X-ray crystallographic studies have shed much light can converge on Smads as a node for signal integration and on the mechanisms of how Smad proteins interact with each thus can positively or negatively influence Smad-mediated other (12). Structurally, Smad proteins consist of two “Mad- transcription (2, 10, 14). Inputs by other pathways occur at homology” domains, MH1 and MH2, which are connected the level of receptor activation, R-Smad complex formation by a flexible linker region. The MH1 domain causes DNA- with Co-Smad4, or at the level of the nuclear translocation binding, whereas the MH2 domain is responsible for of pre-formed R-Smad/Co-Smad complexes (2,