Sequence-specific recruitment of heterochromatin protein 1 via interaction with Krüppel-like factor 11, a human transcription factor involved in tumor suppression and metabolic diseases.

Heterochromatin protein 1 (HP1) proteins are "gatekeepers" of epigenetic gene silencing that is mediated by lysine 9 of histone H3 methylation (H3K9me). Current knowledge supports a paradigm whereby HP1 proteins achieve repression by binding to H3K9me marks and interacting to H3K9 histone methyltransferases (HMTs), such as SUV39H1, which methylate this residue on adjacent nucleosomes thereby compacting chromatin and silencing gene expression. However, the mechanism underlying the recruitment of this epigenetic regulator to target gene promoters remains poorly characterized. In the current study, we reveal for the first time a mechanism whereby HP1 is recruited to promoters by a well characterized Krüppel-like transcription factor (KLF), in a sequence-specific manner, to mediate complex biological phenomena. A PXVXL HP1-interacting domain identified at position 487-491 of KLF11 mediates the binding of HP1α and KLF11 in vitro and in cultured cells. KLF11 also recruits HP1α and its histone methyltransferase, SUV39H1, to promoters to limit KLF11-mediated gene activation. Indeed, a KLF11ΔHP1 mutant derepresses KLF11-regulated cancer genes, by inhibiting HP1-SUV39H1 recruitment, decreasing H3K9me3, while increasing activation-associated marks. Biologically, impairment of KLF11-mediated HP1-HMT recruitment abolishes tumor suppression, providing direct evidence that HP1-HMTs act in a sequence-specific manner to achieve this function rather than its well characterized binding to methylated chromatin without intermediary. Collectively, these studies reveal a novel role for HP1 as a cofactor in tumor suppression, expand our mechanistic understanding of a KLF associated to human disease, and outline cellular and biochemical mechanisms underlying this phenomenon, increasing the specificity of targeting HP1-HMT complexes to gene promoters.

A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth.

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.

Distinct role of Kruppel-like factor 11 in the regulation of prostaglandin E2 biosynthesis.

Kruppel-like factor (KLF) proteins are emerging as key regulators of lipid metabolism, diabetes, and the biosynthesis of immunological cytokines. However, their role in the synthesis of prostaglandins, widely known biochemical mediators that act in a myriad of cell biological processes remain poorly understood. Consequently, in this study a comprehensive investigation at the cellular, biochemical, and molecular levels reveal that KLF11 inhibits prostaglandin E(2) synthesis via transcriptional silencing of the promoter of its biosynthetic enzyme, cytosolic phospholipase A2alpha. Mechanistically, KLF11 accomplishes this function by binding to the promoter via specific GC-rich sites and recruiting the Sin3-histone deacetylase chromatin remodeling complex. Further functional characterization reveals that this function of KLF11 can be reversed by epidermal growth factor receptor-AKT-mediated post-translational modification of threonine 56, a residue within its Sin3-binding domain. This is the first evidence supporting a relevant role for any KLF protein in doing both: transcriptionally inhibiting prostaglandin biosynthesis and its reversibility by an epidermal growth factor receptor-AKT signaling-mediated posttranslational mechanisms.

Rodent endosonography to monitor esophageal cancer.

Animal models of luminal cancers are important to understand and assess chemopreventive and chemotherapeutic interventions. However, the ability to assess tumor growth and response without animal sacrifice is limited. We assessed the ability of luminal sonography to assess the presence of tumor and its size in a surgical esophagojejunostomy model of esophageal cancer. Luminal sonography had a sensitivity of 88%, specificity of 100%, and accuracy of 93% in identifying the esophageal cancers. The tumor dimensions on luminal sonography were within 11% of autopsy measurements. Minimal tumor dimension was 2 mm and maximum 6.2 mm. The procedure was feasible without technical difficulty. In conclusion, rodent endosonography is a useful technique that can accurately determine the presence of tumors as well as their dimensions.

Combinatorial chemoprevention reveals a novel smoothened-independent role of GLI1 in esophageal carcinogenesis.

Reflux-induced injury promotes esophageal adenocarcinoma, one of the most rapidly increasing, highly lethal cancers in Western countries. Here, we investigate the efficacy of a combinatorial chemoprevention strategy for esophageal adenocarcinoma and characterize the underlying molecular mechanisms. Specifically, our approach involves the use of ursodeoxycholic acid (Urso) due to its ability to decrease injury-inducing bile salts in combination with Aspirin to mitigate the consequences of injury. We find that Urso-Aspirin combination reduces the risk of adenocarcinoma in vivo in animals with reflux, decreases the proliferation of esophageal adenocarcinoma cells, and downregulates a key cell cycle regulator, CDK2. Mechanistically, using cell growth, luciferase reporter, expression, and chromatin immunoprecipitation assays, we identify GLI1, a Hedgehog-regulated transcription factor, as a novel target of Urso-Aspirin combination. We show that GLI1 is upregulated during esophageal carcinogenesis, and GLI1 can bind to the CDK2 promoter and activate its expression. Although the Urso-Aspirin combination downregulates GLI1, the GLI1 overexpression not only abrogates the effect of this combination on proliferation but it also restores CDK-2 expression. These findings support that the chemopreventive effect of the Urso-Aspirin combination occurs, at least in part, through a novel GLI1-CDK2-dependent mechanism. To further understand the regulation of CDK2 by GLI1, both pharmacologic and RNAi-mediated approaches show that GLI1 is a transcriptional activator of CDK2, and this regulation occurs independent of Smoothened, the central transducer of the Hedgehog canonical pathway. Collectively, these results identify a novel GLI1-to-CDK2 pathway in esophageal carcinogenesis, which is a bona fide target for effective combinatorial chemoprevention with Urso and Aspirin.

Chemoprevention in Barrett's esophagus.

Barrett's metaplasia-associated esophageal adenocarcinoma is one of the most rapidly increasing cancers in Western countries. Whereas early detection remains the cornerstone of prevention, chemoprevention is emerging as a complementary strategy. Carcinogenesis in Barrett's mucosa is a multistep process in which cellular growth becomes progressively dysregulated. Fortunately, the process of carcinogenesis is a protracted one, which provides ample opportunity for intervention. In this review, we will discuss various potential chemoprevention targets and rationale behind their use to prevent Barrett's related esophageal adenocarcinoma. We will also critically appraise the emerging preclinical and clinical literature regarding prevention of neoplasia in Barrett's esophagus.

Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. It is proposed that one of the possible mechanisms is dedifferentiation of squamous epithelium into columnar epithelium. The pathophysiology through which this metaplasia occurs is unknown. A recent study by serial analysis of gene expression showed that bone morphogenetic protein 4 (BMP-4) is uniquely expressed in BE. In this study, the role of the BMP pathway in the metaplastic transformation of normal squamous cells into columnar cells was examined. METHODS: Tissues from patients with esophagitis and BE and in an esophagitis-BE rat model were examined for the activation of the BMP pathway. Short-term cultures of primary normal squamous esophageal cells were treated with BMP-4, and cell biological changes were examined by Western blot analysis, immunohistochemistry, and microarrays. RESULTS: In both human and rat tissues, the BMP pathway proved to be activated in esophagitis and BE. Upon incubation of squamous cell cultures with BMP-4, the cytokeratin expression pattern showed a shift that was consistent with columnar epithelium. Involvement of the BMP pathway was suggested by up-regulation of Phosphorylated-Smad 1/5/8 (P-Smad 1/5/8) that was effectively blocked by Noggin, a BMP antagonist. Comparison of the gene expression profiles of squamous cells, BMP-4-treated squamous cells, and BE cells showed a significant shift in the profile of the BMP-4-treated squamous cells toward that of the cultured BE cells. CONCLUSIONS: These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.

Modeling the effects of prl mutations on the Escherichia coli SecY complex.

The apparatus responsible for translocation of proteins across bacterial membranes is the conserved SecY complex, consisting of SecY, SecE, and SecG. Prior genetic analysis provided insight into the mechanisms of protein export, as well as the interactions between the component proteins. In particular, the prl suppressor alleles of secE and secY, which allow export of secretory proteins with defective signal sequences, have proven particularly useful. Here, we report the isolation of novel mutations in secE and secY, as well as the phenotypic effects of combinations of prl mutations. These new alleles, as well as previously characterized prl mutations, were analyzed in light of the recently published crystal structure of the archaeal SecY complex. Our results support and expand a model of Prl suppressor activity that proposes that all of the prlA and prlG alleles either destabilize the closed state of the channel or stabilize the open form. These mutants thus allow channel opening to occur without the triggering event of signal sequence binding that is required in a wild-type complex.