BACKGROUND & AIMS: Barrett's esophagus is considered to be a metaplastic lesion that predisposes for esophageal adenocarcinoma. Development of Barrett's esophagus is considered to be driven by sonic hedgehog mediated bone morphogenetic protein (BMP) signaling. We aimed to investigate in preclinical in vivo models whether targeting canonical BMP signaling could be an effective treatment for Barrett's esophagus. METHODS AND RESULTS: Selective inhibition of BMP2 and BMP4 within an in vivo organoid model of Barrett's esophagus inhibited development of columnar Barrett's cells, while favoring expansion of squamous cells. Silencing of noggin, a natural antagonist of BMP2, BMP4, and BMP7, in a conditional knockout mouse model induced expansion of a Barrett's-like neo-columnar epithelium from multi-lineage glands. Conversely, in this model specific inhibition of BMP2 and BMP4 led to the development of a neo-squamous lineage. In an ablation model, inhibition of BMP2 and BMP4 resulted in the regeneration of neo-squamous epithelium after the cryoablation of columnar epithelium at the squamocolumnar junction. Through lineage tracing the generation of the neosquamous mucosa was found to originate from K5thorn progenitor squamous cells. CONCLUSIONS: Here we demonstrate that specific inhibitors of BMP2 and BMP4 attenuate the development of Barrett's columnar epithelium, providing a novel potential strategy for the treatment of Barrett's esophagus and the prevention of esophageal adenocarcinoma.

Summary Introduction Esophageal adenocarcinoma (EAC) is an aggressive cancer, associated with reflux esophagitis and intestinal metaplasia (IM). One underlying biological mechanism, which possibly drives the development of EAC, is the dysregulated expression of Bone Morphogenetic Proteins (BMPs). Aim To investigate if local delivery of Noggin, a BMP antagonist, reduced EAC. Methods After obtaining proof of principal on local delivery of a Noggin/Sucralfate substance, a randomized controlled trial to test the effects of Noggin on EAC development was performed in a surgical rat model. In the model, an esophago-jejunostomy leads to development of reflux-esophagitis, IM and eventually EAC. Rats were treated by Noggin/Sucralfate or Sucralfate alone. Treatment was administered from 26 to 29 weeks after the operation. Results Of the 112 operated rats, 52 survived beyond 26 weeks. Finally, 25 rats treated with Noggin/Sucralfate and 21 with Sucralfate, were evaluated. At the end, 39 (85%) of the animals had IM while 28 (61%) developed cancer. There were significantly more cancers in the Noggin/Sucralfate arm (50%) versus the Sucralfate group (73%) (Chi square, P < 0.05). Most cancers were mucous producing T3 adenocarcinomas. There were no significant differences in the amount of IM, size or grade of the cancers, or expression of columnar and squamous markers between the two groups. Conclusion In this study, we demonstrated that inhibition of BMPs by Noggin reduced development of EAC in a surgical esophagitis-IM-EAC rat model. In future, effective targeting of the BMP pathway with selective BMP-inhibitors could become an important asset to improve EAC patient outcome.

Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett's esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due to lack of effective eradication therapies. The shortage of innovative treatment options is mostly attributable to the paucity of adequate in vivo models of neo-columnar epithelium regeneration. This protocol describes the generation of a cryoablation model to study regeneration of neo-epithelia at the SCJ. Cryoablation of the columnar and squamous mucosa at the SCJ was achieved through local application of liquid N2O in wild-type and reporter mice in combination with acid suppression. Acid suppression alone, showed restoration of the SCJ with normal histological features of both the neo-columnar and neo-squamous epithelium within 14 days. As a proof of principle, mice were treated with mNoggin, an inhibitor of bone morphogenetic proteins (BMPs), which are involved in the development of columnar epithelia. Local application of mNoggin to the ablated area at the SCJ significantly reduced the development of the neo-columnar mucosa. Although this model does not faithfully recapitulate the exact characteristics of Barrett's esophagus, it is a well-suited tool to study the mechanisms of therapeutic inhibition of neo-columnar regeneration. It therefore represents an efficient and easy platform to test novel pharmacological therapies for treatment of neo-epithelial lesions at the SCJ.

Background: Gut microbiota in early life plays a vital role in intestinal development of the offspring.Our previous study has reported that neonatal Lactobacillus rhamnosus GG (LGG) colonization could promote the intestinal development, which might show long-term positive effects on the heath of the host.However, whether LGG colonization in early life could inhibit the intestinal tumor formation of offspring in adulthood or not remains unknown.Method Adult C57BL/6 female mice were mate with Apc min/+ male mice, and checked daily.Pregnant mice with the same conception time were given 10 8 cfu LGG live bacteria (LGG colonization group, LC group) or LGG fixed bacteria (LGG non-colonization group, NC group) from the 18th day after pregnancy until natural birth.Apc min/+FCCC offspring was chosen after genotyping.Then the progeny mice were given by gavaged once a day in 0-5 days of birth with 10 7 cfu of LGG live bacteria or LGG fixed bacteria, respectively.The body weight of offspring mice was recorded.The intestinal development, mucosal barrier, mucosal inflammation, and microbiota of half 3-week old offspring were assessed.Meanwhile, after weaning the remaining progeny mice were fed normal diet until 12-week old, and the multiplicity of intestinal tumor, the tumor cell proliferation and apoptosis, Wnt signaling pathway and gut microbiota in 12-week old adulthood were also analyzed.Result LGG colonization can be detected only in the feces of offspring in LC group from childhood to adulthood.The body weight between two groups was similar at the first week, while the offspring mice of the LC group were heavier at 2-12 weeks compared with NC group.We found that LGG colonization in early life significantly promote the intestinal development and protected barrier function in 3-week old progeny mice.LGG colonization decreased intestinal pro-inflammatory cytokines production indicated that low-grade inflammation was inhibited, while no obvious microscope inflammation in both groups at 3-week old was observed by H&E staining.Interestingly, the composition and diversity of gut microbiota of 3-week old offspring were significant different between two groups, even after 9 weeks' consumption of normal diet, the microbiota composition in adulthood was still different.LC group characterized by increased abundance of Mollicutes and Eisenbergiella and reduction of Verrucomicrobiales, Akkermansia and Prevotella.Ultimately, LGG colonization could inhibits adenoma cell proliferation and promotes apoptosis.Meanwhile LGG inhibited intestinal tumor formation through regulation of the tumor associated Wnt signaling pathway in adulthood.Conclusion: Intestinal colonization with LGG in early life could promote the intestinal development, protect the barrier function and regulate gut microbiota, therefore, inhibit the tumor formation in adulthood of Apc min/+FCCC mice.

Bile acid reflux is known to be associated with the development of Barrett’s esophagus and esophageal adenocarcinoma (EAC), yet the role of specific bile acids and the mechanism behind the metaplastic changes is unclear. Here, we demonstrate that multi-layered glandular structures at the squamo-columnar junction in mice contain multiple cell lineages, which resemble the human esophageal submucosal gland ducts. Exposing mice to patient’s refluxates induced expansion of multi-layered glandular structures and development of columnar metaplasia at the squamo-columnar junction. The glycine conjugated bile acids induced an intestinal type of metaplasia more typical for Barrett’s esophagus. Through lineage tracing, we excluded the involvement of K5+, DCLK1+, and LGR5+ progenitor cells as the primary source in the development of the glandular metaplastic epithelium. We show that the mechanism behind development of metaplasia involves crypt fission and may be independent of stem cell proliferation. Our findings support the hypothesis that in humans, BE arises from non-squamous cells residing in submucosal gland ducts and that induction of intestinal type of metaplasia is most effectively induced by glycine-conjugated bile acids. These novel insights may lead to more effective strategies to prevent development of Barrett’s esophagus and esophageal adenocarcinoma.

Abstract Background In Western countries, the highly malignant Esophageal adenocarcinoma (EAC) have the most dramatically rising incidence of all malignancies. BMP4 is a growth factor important for carcinogenesis. We found that BMP4 is aberrantly expressed in Barret's esophagus, the pecursor lesion of EAC, and that together with CDX2 drives the intestinalization of epithelial metaplasia. However, its role in esophageal adenocarcinoma (EAC) remains uncertain. Methods Method: To elucidate whether BMP4 is involved in malignancy in EAC we used an RNA sequencing database of 56 EAC treatment naive endoscopic biopsies to investigate if there is a subgroup of cancers with high BMP signaling. We validated results by qPCR and immunohistochemistry in matching tumor samples. Next we used our recently developed effective and highly specific anti-BMP4 antibodies(1,2) to study the effect of inhibition of BMP4 on both in vitro as well as in vivo models of EAC. Results Using a gene set that was recently published for BMP signaling, we were able to distinguish a subgroup of EAC patients with increased BMP signaling. By IHC we confirmed that 70% of EAC tumors express BMP4 at the protein level. We found that patients with high levels of BMP4 expression tend to have a poorer recurrence-free survival compared to patients with low BMP4 expression, which suggests a more aggressive tumor behavior in BMP4 expressing EAC tumors. Most importantly, inhibition of BMP4 function in EAC cells by our recently developed anti-BMP4 antibodies lead to an increase in chemo-sensitivity and a decreased in invasive and migratory capabilities in vitro. Preclinical in vivo studies with a patient-derived tumor xenograft mouse model of an EAC tumor confirmed that anti-BMP4 antibodies can effectively reduce tumor growth and synergistically act with chemotherapy agents. Conclusion We identified a subgroup of EAC with increased BMP signaling. Our studies support a role of BMP4 in chemo-resistance and invasiveness in EAC, and indicate that inhibition of BMP4 with highly our specific llama-derived antibodies is an attractive therapy for improving outcomes of EAC. Disclosure All authors have declared no conflicts of interest.

significantly enhanced the sensitivity of cells to epirubicin (IC50=0.594µM).Moreover, pharmacologic inhibition of AXL with R428 significantly sensitized FLO-1 cells to epirubicin (IC50=0.77µM).The Annexin V/DAPI staining and FACS analysis data indicated that AXL overexpression in OE33 cells decreased apoptosis events by 61.2% relative to control in response to epirubicin.Conversely, knockdown of endogenous AXL in FLO-1 cells significantly increased apoptosis by 33% relative to control in response to epirubicin.The changes in apoptosis levels in both cell models were confirmed by Western blot analysis of molecular markers of apoptosis (caspase-3 and PARP).To determine the mechanism of epirubicin resistance, we evaluated the effect of AXL on the expression of c-MYC.The real-time PCR and Western blot data showed that overexpression of AXL up-regulated c-MYC mRNA and protein levels in OE33 cells.Conversely, knockdown of AXL in FLO-1 cells decreased c-MYC mRNA and protein expression.In addition, blocking of c-MYC activity by the specific inhibitor 10058-F4 remarkably sensitized the FLO-1 cells to epirubicin.Mechanistic investigations indicated that AXL upregulates c-MYC expression and activity through activation of the AKT/b-catenin pathway.Data from a tumor xenograft mouse model showed that pharmacologic inhibition of AXL with a sub-lethal dose of R428 (10 mg/kg) synergistically sensitizes resistant FLO-1 cells to epirubicin.Conclusions: Our data demonstrate that AXL promotes epirubicin resistance through transcriptional upregulation of c-MYC in EAC.We propose targeting AXL with R428 as a novel therapeutic approach to overcome epirubicin resistance in tumors with AXL overexpression and c-MYC activation.

Abstract 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.

Surveillance of Barrett’s oesophagus allows us to study the evolutionary dynamics of a human neoplasm over time. Here we use multicolour fluorescence in situ hybridization on brush cytology specimens, from two time points with a median interval of 37 months in 195 non-dysplastic Barrett's patients, and a third time point in a subset of 90 patients at a median interval of 36 months, to study clonal evolution at single-cell resolution. Baseline genetic diversity predicts progression and remains in a stable dynamic equilibrium over time. Clonal expansions are rare, being detected once every 36.8 patient years, and growing at an average rate of 1.58 cm 2 (95% CI: 0.09–4.06) per year, often involving the p16 locus. This suggests a lack of strong clonal selection in Barrett’s and that the malignant potential of ‘benign’ Barrett’s lesions is predetermined, with important implications for surveillance programs.

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.

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.

Barrett's 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.

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 (783.8METmin/week for BE cases vs. 826.2for 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.

The molecular mechanisms leading to epithelial metaplasias are poorly understood. Barrett's 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.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the animal reflux-inflammation models for Barrett's 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 Barrett's adenocarcinoma; statins in chemoprevention and treatment of esophageal cancer; and biomarkers as potential targets in Barrett's adenocarcinoma.