Cathrine J. DeMars

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

Background: Chromatin remodeling mechanisms utilized by Krüppel-like factor proteins remain poorly defined. Results: Krüppel-like factor 11 directly recruits heterochromatin protein 1α to promoters in a sequence-specific manner. Conclusion: Coupling to heterochromatin protein 1 α and its histone methyltransferase underlies Krüppel-like factor-mediated gene expression and tumor suppression. Significance: This data advances our understanding of how chromatin-mediated mechanisms achieve these functions with increased specificity for target genes. 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.

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.

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 E2 synthesis via transcriptional silencing of the promoter of its biosynthetic enzyme, cytosolic phospholipase A2α. 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.

Endoscopic appearance and extent of high-grade dysplasia in Barrett’s esophagus can predict the presence of cancer at esophagectomy

Endoscopic appearance and extent of high-grade dysplasia in Barretts esophagus can predict the presence of cancer at esophagectomy

Sa1965 Garcinol Interferes With Oncogenic IL1b-STAT3 and Facilitates Tumor Suppressive KLF11-Sin3A to Down-Regulate AKT1 Expression and Cell Growth in Barrett's Epithelium

ESR2 physical interactions. Surprisingly, our preliminary results showed an upregulation in EGFR signals in ERB041 treated ESR2 transfectants. Summary: In AA with adenomas, reductions in ESR2 expression might contribute to higher colon cancer incidence and worse prognosis. The in vitro studies confirmed that ESR2 suppresses colon cancer cell proliferation. We postulate that the antiproliferative effects of ESR2 involve miR-145 dependent, EGFRindependent pathways. Our study suggests that ESR2 down-regulation might play an important role in colon cancer risk and contribute to racial differences in cancer progression.

388 IL-1β-STAT3 Mediated Up-Regulation of AKT1 Expression in Barrett's Esophagus: A Potential Link Between Inflammation and Carcinogenesis

BACKGROUND AND AIM: We have recently shown that, in addition to AKT1 activation through its phosphorylation, overall increased expression of AKT1 that is observed during neoplastic progression in patients with Barretts also plays a direct role in this injury-induced oncogenesis. Since pro-inflammatory cytokine IL-1β promotes esophageal adenocarcinoma and its downstream effector STAT3 could bind to the core promoter region of AKT1, it is plausible that IL-1β-STAT3-AKT1 could link inflammation to carcinogenesis in Barretts esophagus. The aims of this study were to examine IL-1 β-STAT3 mediated regulation of AKT1 expression and explore the underlying mechanisms in Barretts epithelium. METHODS and RESULTS: Using Barretts epithelial cells, we noted that both IL-1 β and constitutively active STAT3c induced AKT1 promoter activity. Moreover, in a dose and time dependent manner IL-1β increased AKT1 mRNA expression. Similarly, constitutively active STAT3c also increased AKT1 mRNA expression. We also noted that IL-1 β increased cellular p300 levels, a histone acetyl transferase that STAT3 recruits on target promoter chromatin to activate them by acetylating histone H3. We found that while constitutively active STAT3c increased AKT1 promoter activity/expression, there was an increased acetylation of AKT1 promoter (ChIP using anti H3 K18 and 27 antibody) during neoplastic progression in Barretts epithelium. Additionally, both IL-1β and STAT3c partially interfered with repression of AKT1 promoter by anti-inflammatory transcription factor KLF11, a known recruiter of Sin3-HDAC that deacetylates and represses AKT1 promoter. Together, these findings raise a possibility that pro-inflammatory IL-1β, by simultaneously increasing p300 and STAT3 activation could direct p300 onto the AKT1 promoter to hyper acetylate and activate AKT1 promoter. Alternatively, pro-inflammatory IL-1β-pSTAT3 through antagonizing the antiinflammatory KLF11-Sin3-histone deacetylase pathway preserve AKT1 promoter acetylation to activate AKT1 promoter/expression. CONCLUSION: Our observations expose an antagonism between pro-inflammatory IL-1β-STAT3-p300 and anti-inflammatory KLF11-Sin3HDAC pathways in regulating oncogenic AKT1 during neoplastic transformation in Barretts esophagus, providing a potential link between inflammation and carcinogenesis.

407a Oncogenic AKT1 Regulation by Tumor Suppressor KLF11: Implications in Growth Regulation During Neoplastic Transformation in Barrett's Esophagus and Interference by Carcinogenic Bile Acid Dependent Erk2 and PKCa Activation

Pancreatic adenocarcinoma is the fifth leading cause of cancer death, with an abysmal 5year survival rate of less than 5%, due in large part to the high incidence of metastasis. Metastatic invasion represents a complex synergy of cell motility, adhesion, and matrix remodeling. How these multiple processes are coordinated during dissemination is a central focus of tumor cell biology. The small oncogenic G protein Rac1 is a major regulator of cell motility, and it is activated through the action of guanine nucleotide exchange factors (GEFs). A critical GEF in tumor cell biology is the proto-oncogene Vav1, which is ectopically expressed in most pancreatic tumors to cause poor prognosis in patients and enhanced tumor growth In Vitro. Vav1 interacts directly with the large GTPase Dynamin 2 (Dyn2), though the function of this interaction is not known. Importantly, Dyn2 is also markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. We have recently found that Dyn2 binds Vav1 directly in pancreatic tumor cells. Therefore, the GOAL of this study was to test if the Dyn2Vav1 interaction could promote the activation of Rac1 and the invasive migration of pancreatic tumor cells. RESULTS: We found that disruption of the Dyn2-Vav1 interaction impairs Rac1 activation and subsequent lamellipodia formation and cell migration, indicating that Dyn2-Vav1 binding regulates tumor cell motility. Surprisingly, RNAi-mediated suppression of Dyn2 or disruption of Dyn2-Vav1 binding results in a dramatic reduction in Vav1 protein stability, resulting in diminished levels of this proto-oncogene and decreased Rac1 activation and migration. Remarkably, this degradative reduction is a result of Vav1 targeted transport to the lysosome, as treatment of these cells with the protease inhibitor chloroquine prevents Vav1 loss, and immunofluorescent cell staining shows a dramatic localization of Vav1 to the lysosomal lumen when it cannot bind Dyn2.CONCLUSION: These findings demonstrate a novel regulatory role for direct Dyn2 binding in Rac1 activation, lamellipod protrusion and cellular migration through the stabilization of Vav1, and demonstrate a potent mechanism by which metastatic migration is upregulated in pancreatic tumor cells. This study was funded by NCI CA104125 to MAM.

Su1051 Dietary Macronutrients Alter Erk2-AKT1-PGE2 Pathway in Barrett's Mucosa: Potential Role During Carcinogenesis in Barrett's Epithelium

Background: No-shows and appointment cancellations are especially salient to gastroenterology. Data shows that the incidence of colorectal cancer is down-trending, which correlates to increased colorectal cancer awareness and increase in the number of per capita screening colonoscopies. Patients lost to follow-up because of a no-show or cancellation (NS/C) might be at a higher risk of developing malignancy because of a missed screening opportunity. If particular patient demographics can be identified that are associated with NS/C, specific interventions can be proposed to capture those patients at possible increased risk. Aim: To determine if certain patient demographics contribute to the NS/C. Methods: A retrosepctive chart review using the Computerized Patient Records System (CPRS) at the Carl T.Hayden VA Medical Center in Phoenix, Arizona analyzed 17 weeks of data regarding NS/C. Data was obtained regarding the patients: age, gender, new vs. follow appointment, type of procedure vs. clinic appointment, reason for appointment, and origin of the appointment, whether within the GI department or through open access primary care consultation. Results: 17 weeks of data were reviewed. A majority were male (92.2%). Ages ranged from 23-88 years and average age was 57.5 years old. 14.8% of patients were younger than 50. More NS/C were for follow-ups (55.8%) rather than new consults (44.2%). There were also more NS/C for colonoscopies (73.8%) than EGDs (24.1%) and paracentesis contributed to only 1.9% of the total. Appointments originating from outside the GI department were more likely to result in NS/C as 73.6% of those appointments originated from the PCP. Only 26.1% of NS/C originated from within the GI department. A specific analysis of coloscopy was made (table) revealing that 265 colonoscopies were performed. 19.3% of the NS/C colonoscopies scheduled for symptomatic complaints (pain, diarrhea, constipation, hematochezia) came from within the GI department while 80.7% came from the patients PCP. This was supported by an overwhelming majority of /C screening and surveillance appointments that also came from outside the GI department (87.1%). Conclusions: This data illustrates that of patients who fail to keep their appointments, they are likely to be older than 50, with asymptomatic complaints, who are referred to the GI department by their PCPs. This is valuable information because patients lost in the NS/C process represent a missed opportunity for possible positive interventions including but not limited to the prevention of colorectal cancer. By identifying these variables, steps can be taken to minimize their contribution to the NS/C rate in the future.

Molecular determinants of metaplastic and neoplastic transformation in the esophageal epithelium.

A metaplastic change where normal squamous mucosa of the esophagus is replaced by columnar mucosa is considered an adaptive process to withstand the chronic gastroesophageal reflux injury. However, this phenotypic switch in the esophageal mucosa is associated with an increased propensity to undergo neoplastic transformation. In this chapter, we review the molecular alterations that are relevant to metaplastic and neoplastic transformations in the esophagus and also discuss the mechanisms that could potentially contribute to this transformation.