Danielle Straub

A pSMAD/CDX2 Complex Is Essential for the Intestinalization of Epithelial Metaplasia

The molecular mechanisms leading to epithelial metaplasias are poorly understood. Barretts esophagus is a premalignant metaplastic change of the esophageal epithelium into columnar epithelium, occurring in patients suffering from gastroesophageal reflux disease. Mechanisms behind the development of the intestinal subtype, which is associated with the highest cancer risk, are unclear. In humans, it has been suggested that a nonspecialized columnar metaplasia precedes the development of intestinal metaplasia. Here, we propose that a complex made up of at least two factors needs to be activated simultaneously to drive the expression of intestinal type of genes. Using unique animal models and robust in vitro assays, we show that the nonspecialized columnar metaplasia is a precursor of intestinal metaplasia and that pSMAD/CDX2 interaction is essential for the switch toward an intestinal phenotype.

Strategy for prevention of cancers of the esophagus

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the animal reflux–inflammation models for Barretts esophagus and esophageal adenocarcinoma; genomic/epigenomic analyses; eflornithine‐based combinations; the molecular derangements that promote neoplastic transformation; the role of COX‐2 inhibitors, proton pump inhibitors, and phase II trials in Barretts adenocarcinoma; statins in chemoprevention and treatment of esophageal cancer; and biomarkers as potential targets in Barretts adenocarcinoma.

Active matrix metalloproteases are expressed early on and are high during the Barrett's esophagus malignancy sequence

Abstract Objective. Molecular processes underlying Barrett’s malignant development are poorly understood. Matrix metalloproteases (MMPs) are enzymes involved in inflammation, tissue remodeling, and malignant development. Therefore, active MMPs may have a role in early metaplasia development and Barrett’s esophagus’ malignant progression. We desired to gain more insight into the role of MMPs during the Barrett’s esophagus pathogenesis sequence. Material and methods. In a surgical Barrett’s mouse model, and in nonmalignant Barrett’s and malignant esophageal cell lines, the activity of MMPs was investigated using a MMP activatable probe. MMP activity was further validated in Barrett’s esophagus and esophageal adenocarcinoma patient biopsies and was further differentiated by investigating MMP9 and MMP13 expressions. Results. The mouse model showed probe activation in stromal cells early on in the esophagitis and metaplasia stages. MMP probe activation was higher in the Barrett’s and cancer cell lines and biopsies as compared to normal cells and tissues. Co-immunostainings confirmed that, at the tissue level, the probe activation was mostly confined to CD45-positive stromal cells. MMP13 expression was highest in Barrett’s metaplasia, whereas MMP9 was highest in the esophageal adenocarcinomas. Conclusion. During the Barrett’s pathogenesis process, MMP activity is increased early on in the inflamed esophagus and remains high in metaplasia and esophageal adenocarcinoma. However, there is a switch of MMP13 to MMP9 expression once neoplasia develops. In the future, detecting specific MMP subtypes could be used for distinguishing nonmalignant from neoplastic Barrett’s esophagus.

Increased phosphorylation on residue S795 of the retinoblastoma protein in esophageal adenocarcinoma

Due to its increasing incidence and relatively poor prognosis, esophageal adenocarcinoma (EAC) is becoming a significant health problem. Elucidating the mechanisms underlying EAC development is of great importance to improve upon current conventional treatment strategies. Insight into phosphorylation has proven to be useful for the development of diagnostic and molecular treatment strategies in cancer. A pathway largely dependent on phosphorylation and frequently deregulated in cancer is the cell cycle regulating p16-retinoblastoma (Rb) pathway. We investigated kinase activity, specifically phosphorylation within the p16-Rb pathway, in EAC. A high-throughput peptide tyrosine kinase array containing short peptides representing 100 proteins with known phosphorylation sites, was used to assess phosphorylation activity in EAC. Also, specific phosphorylation changes of the cell cycle protein Rb and its upstream regulator P16 were validated through immunoblotting in EAC and normal esophageal cells and tissues. Phosphorylation activity was higher in EAC tissues as compared to normal squamous esophageal tissues. A majority of the proteins significantly higher phosphorylated in EAC were found to be involved in cell structure maintenance and immunity. Validation of Rb phosphorylation in EAC biopsy specimens and cell lines showed hyper phosphorylation of Rb associated with aberrant P16 expression in the cancer tissues. The specific Rb (S795) residue was significantly higher phosphorylated in EAC compared to normal esophageal tissue (Wilcoxon paired rank test, p=0.004). Investigation of Rb (S795) phosphorylation may indicate targets for intervention and give more molecular insight in EAC.

Abstract C04: In vivo upregulation of bone morphogenic protein 4 (BMP4) in esophageal cancer is through tumor-stroma crosstalk

Bone morphogenic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor-beta (TGFβ;) superfamily. Amongst them, BMP4 is becoming increasingly attractive due to its crucial role in the development of many cancers. Whereas in malignancies such as glioblastoma and myeloma high levels of BMP4 are associated with benign features, in gastrointestinal cancers BMP4 possesses tumorigenic capacities. In these cancers, BMP4 acts on tumor epithelial cells enhancing their pro-metastatic behavior and chemo-resistance. However, whether BMP4 is being secreted by the epithelial cells or by the stroma, has not been well established yet, in part due to a lack of specific anti-BMP4 antibodies. To elucidate the functional implications of the origin of BMP4 production, we made use of a novel esophageal adenocarcinoma (EAC) tumor xenograft model and a newly generated Llama-derived anti-BMP4 antibody. Using in vivo molecular imaging techniques we find that BMP4 expression in the tumor is increased after engraftment of BMP4 negative human EAC tumor cells into immunodeficient mice. Immunohistochemical studies of the engrafted tissue reveal both a mouse and a human origin of BMP4 protein, suggesting that both epithelial as well as stromal cells are involved in BMP4 secretion. This implies that whereas the stroma directly secretes BMP4; it also produces molecules that activate autocrine BMP4 production by the cancer epithelial cells. In vitro analysis such as co-cultures with different stromal cells, revealed the functional effects of stroma-derived BMP4 on tumor cells, and how our anti-BMP4 antibodies can inhibit these. Further, these studies also uncovered the stromal components that induce BMP4 secretion by the human epithelial cells. In sum, these studies suggest a pivotal role of the microenvironment in regulating both directly and indirectly BMP4 expression in a model of esophageal cancer. As BMP4 has also been shown to be responsible for tumor progression in other gastrointestinal cancers, stromal regulation of BMP4 production might not be restricted to EAC, and might be a common feature in gastrointestinal cancers. Finally, as our anti-BMP4 Llama-derived antibodies can inhibit these effects in vitro, they might represent a novel therapy in targeting stromal function and preventing metastasis. Citation Format: Silvia Calpe, Matthew Read, Maria del Carmen Sancho-Serra, Danielle Straub, Nick Clemons, David Liu, Wayne Phillips, Kausilia K. Krishnadath. In vivo upregulation of bone morphogenic protein 4 (BMP4) in esophageal cancer is through tumor-stroma crosstalk. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C04.

542 Multi Layered Columnar Epithelium (MLCE) Induced by Bile At the Squamo-Columnar Junction in Mice Originates From Squamous and Columnar Cells

Barretts esophagus (BE), a premalignant condition of esophageal adenocarcinoma (EAC) is defined as the replacement of normal stratified squamous epithelium of the lower esophagus by metaplastic columnar epithelium (Spechler 2014). The observation of multi-layered columnar epithelium regularly observed at the squamo-columnar junction (SCJ) in BE patients suggests a transitional stage between these two types of epithelia. Studies, in both human and rats, have shown that especially the combination of acid and biles can induce multilayered columnar epithelium (MLCE) and metaplasia at the SCJ (Vaezi 1995, Chen 2008, Glickman 2009). In this study we investigated if the diverse layers as observed in MLCE is a transitional stage between epithelia originating from one progenitor cell, or is a mix of different types of epithelia with diverse progenitors. 0.5% deoxycholic acid (DCA) was given to mice in drinking water for a maximum of 30 weeks. Animals were sacrificed and studied by histology and immunohistochemistry (IHC) at different time points. The combination of bile and acid induced gland formation at the SCJ starting from week 10. The MLCE glands were positive in the outer layer for squamous markers K14, p63 and K5 and in the inner layer for columnar markers K19, TFF2 and Alcian Blue. In one of our previous studies we also observed Lgr5 positive cells (RNA in situ) in these multi-layered glands (Mari et al. 2014). To investigate if the multi-layered epithelium originates from a squamous or columnar stem cell, we performed lineage tracing experiments. A Cytokeratin 5 (K5)-cre (n=20) mouse specific for tracing squamous progenitor lineages, and a LeucineG-coupled receptor (Lgr5)-cre mouse (n=20) were crossed with Rosa-lacZ mice. Lineage tracing was induced by tamoxifen injection prior to bile treatment at the age of 8 weeks. Mice were sacrificed 15 weeks after induction. K5-lacZ positive cells were present in the outer layer of the gland demonstrating that the origin of these cells is from squamous progenitors, which is in concordance with the IHC results. Surprisingly, the columnar inner layer of the gland was devoid of K5and Lgr5-lacZ positive cells, suggesting that the origin cell for this layer is neither K5 or Lgr5. Negative K5 lineage tracing in columnar cells within the MLCE in mice excludes the transdifferentiation hypothesis in which squamous cells change into columnar cells, which has been a prevailing theory for many years. The negative lineage experiments for LGR5 suggests a columnar cell of origin, other than Lgr5 for the inner layer. Continuous acid and bile reflux changes the environment at the SCJ in mice and disrupts the natural homeostatic environment of squamous and columnar cells. It appears that MLCE is the result of competitive interactions between cell lineages driven by environmental changes.

Sa1836 Active Metalloproteases Are Expressed Early on During the Esophagitis-Metaplasia Sequence and Are High in Barrett's Esophagus and Esophageal Adenocarcinoma

G A A b st ra ct s 3 intensity levels (walking, moderate intensity and vigorous intensity) during the past 7 days. We categorized level of physical activity by low, moderate, or high using IPAQ definitions. We also estimated metabolic equivalent minutes per week (MET-min/wk) by weighting the reported minutes/week within each activity category by a MET energy expenditure estimate for each category of activity. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) using multivariable logistic regression models adjusting for age, sex, race, GERD symptoms, H. pylori, BMI and high waist-to-hip ratio. Results: There were 323 cases with BE and 1849 controls (1347 from endoscopy and 502 from the primary care clinic) (Table 1). Most (68.8%) patients were in the lowest category of physical activity, 13.5% had moderate activity and 11.2% had high activity (6.5% were missing). BE cases were more likely to be in the high category physical activity category than controls (13.6% vs. 10.8% p=0.08). The overall average MET-min/week for walking were 909 for BE cases vs. 561 in controls (p=0.16); with similar findings for those with moderate activity (1094 METmin/week for BE cases vs. 755 for controls, p=0.175), and for vigorous activity (783.8 METmin/week for BE cases vs. 826.2 for controls, p=0.927). In multivariable logistic regression, physical activity was not significantly associated with BE (OR for high vs. low physical activity: 1.19 (95%CI: 0.8-1.73). In fact, BE patients were more likely to have high level of physical activity than PCP controls (OR: 2.1; 95% CI: 1.17-3.6). Conclusions: Recent amount and intensity of physical activity does not seem to be associated with significant changes in the risk of BE. Studies are required examine long term effects of physical activity.