Kelly A. Whelan

In Vivo Modulation of O-GlcNAc Levels Regulates Hippocampal Synaptic Plasticity through Interplay with Phosphorylation

O-Linked N-acetylglucosamine (O-GlcNAc) is a cytosolic and nuclear carbohydrate post-translational modification most abundant in brain. We recently reported uniquely extensive O-GlcNAc modification of proteins that function in synaptic vesicle release and post-synaptic signal transduction. Here we examined potential roles for O-GlcNAc in mouse hippocampal synaptic transmission and plasticity. O-GlcNAc modifications and the enzyme catalyzing their addition (O-GlcNAc transferase) were enriched in hippocampal synaptosomes. Pharmacological elevation or reduction of O-GlcNAc levels had no effect on Schaffer collateral CA1 basal hippocampal synaptic transmission. However, in vivo elevation of O-GlcNAc levels enhanced long term potentiation (LTP), an electrophysiological correlate to some forms of learning/memory. Reciprocally, pharmacological reduction of O-GlcNAc levels blocked LTP. Additionally, elevated O-GlcNAc led to reduced paired-pulse facilitation, a form of short term plasticity attributed to presynaptic mechanisms. Synapsin I and II are presynaptic proteins that increase synaptic vesicle availability for release when phosphorylated, thus contributing to hippocampal synaptic plasticity. Synapsins are among the most extensively O-GlcNAc-modified proteins known. Elevating O-GlcNAc levels increased phosphorylation of Synapsin I/II at serine 9 (cAMP-dependent protein kinase substrate site), serine 62/67 (Erk 1/2 (MAPK 1/2) substrate site), and serine 603 (calmodulin kinase II site). Activation-specific phosphorylation events on Erk 1/2 and calmodulin kinase II, two proteins required for CA1 hippocampal LTP establishment, were increased in response to elevation of O-GlcNAc levels. Thus, O-GlcNAc is a novel regulatory signaling component of excitatory synapses, with specific roles in synaptic plasticity that involve interplay with phosphorylation.

ErbB2 requires integrin α5 for anoikis resistance via Src regulation of receptor activity in human mammary epithelial cells

ErbB2, a receptor tyrosine kinase highly expressed in many tumors, is known to inhibit apoptotic signals. Overexpression of ErbB2 causes anoikis resistance that contributes to luminal filling in three-dimensional mammary epithelial acinar structures in vitro. Given that integrins and growth factor receptors are highly interdependent for function, we examined the role of integrin subunits in ErbB2-mediated survival signaling. Here, we show that MCF-10A cells overexpressing ErbB2 upregulate integrin α5 via the MAP-kinase pathway in three-dimensional acini and found elevated integrin α5 levels associated with ErbB2 status in human breast cancer. Integrin α5 is required for ErbB2-mediated anoikis resistance and for optimal ErbB2 signaling to the Mek-Erk-Bim axis as depletion of integrin α5 reverses anoikis resistance and Bim inhibition. Integrin α5 is required for full activation of ErbB2 tyrosine phosphorylation on Y877 and ErbB2 phosphorylation is associated with increased activity of Src in the absence of adhesion. Indeed, we show that blocking elevated Src activity during cell detachment reverses ErbB2-mediated survival and Bim repression. Thus, integrin α5 serves as a key mediator of Src and ErbB2-survival signaling in low adhesion states, which are necessary to block the pro-anoikis mediator Bim, and we suggest that this pathway represents a potential novel therapeutic target in ErbB2-positive tumors.

Hypoxia Suppression of Bim and Bmf Blocks Anoikis and Luminal Clearing during Mammary Morphogenesis

Hypoxia can regulate many cellular processes. We show that hypoxia, via hypoxia-inducible factor (HIF)-1, blocks anoikis of epithelial cells by activating signaling pathways that inhibit expression of proapoptotic proteins Bim and Bmf. Hypoxia also disrupts mammary morphogenesis and blocks anoikis associated with lumen formation in three-dimensional in vitro model of mammary acini.

The oncogene HER2/neu (ERBB2) requires the hypoxia-inducible factor HIF-1 for mammary tumor growth and anoikis resistance.

Background: Oncogene HER2 (ERBB2) regulates breast cancer growth and anoikis resistance. Results: ERBB2 requires hypoxia-inducible factor (HIF)-1 for breast cancer growth in vivo and anoikis resistance in vitro. Conclusion: HIF-1 plays a critical role in ERBB2-positive breast cancers. Significance: Genes co-regulated by ERBB2 and HIF-1 may be novel therapeutic targets for treating breast cancer. ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.

Mechanisms of Barrett's oesophagus: Intestinal differentiation, stem cells, and tissue models

Barretts oesophagus (BE) is defined as any metaplastic columnar epithelium in the distal oesophagus which replaces normal squamous epithelium and which predisposes to cancer development. It is this second requirement, the predisposition to cancer, which makes this condition both clinically highly relevant and an important area for ongoing research. While BE has been defined pathologically since the 1950s (Allison and Johnstone, Thorax 1955), and identified as a risk factor for esophageal adenocarcinoma since the 1970s (Naef A.P., etxa0alxa0J Thorac Cardiovasc Surg. 1975), our understanding of the molecular events giving rise to this condition remains limited. Herein we will examine what is known about the intestinal features of BE and how well it recapitulates the intestinal epithelium, including stem identity and function. Finally, we will explore laboratory models of this condition presently in use and under development, to identify new insights they may provide into this important clinical condition.

Surviving without oxygen: hypoxia regulation of mammary morphogenesis and anoikis.

Tumor hypoxia correlates with resistance to chemotherapy, increased incidence of metastasis and poor clinical prognosis. Early breast cancer lesions, such as carcinoma in situ, characterized by filled lumens, are often associated with hypoxic markers. However, the contribution of hypoxia to changes in tissue architecture in early pre-malignant lesions is not well defined. Using three-dimensional basement membrane cultures of mammary epithelial cells, we recently reported that acini-like structures exposed to hypoxia display epithelial disorganization and delayed lumen formation. We found that hypoxia, via HIF-1α, targets signaling pathways, specifically the Erk-Bim axis, to suppress anoikis-cell death in a 3D acinar model. Here, we discuss these findings further and present additional data, indicating that hypoxia-mediated alterations in acinar architecture and anoikis-associated Erk-Bim signaling are maintained in mammary epithelial cells after reoxygenation. Taken together, these findings may offer new insight into the contribution of hypoxia-mediated signaling in the progression of early breast cancer lesions and possible treatment of hypoxic cancers.

ATG7 Gene Expression as a Novel Tissue Biomarker in Eosinophilic Esophagitis.

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Three-Dimensional Organoids Reveal Therapy Resistance of Esophageal and Oropharyngeal Squamous Cell Carcinoma Cells

Background & Aims Oropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo. We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas. Methods We generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo. Results Tumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients (P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment. Conclusions The single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.

O-010 Novel Regulation of Autophagy and Intestinal Homeostasis Via mRNA Binding Protein IMP1.

Background:IMP1 (Insulin-like growth factor-2 mRNA binding protein 1) is essential for normal gut development and aberrant overexpression promotes colorectal tumors; however, the role of IMP1 in epithelial homeostasis in the adult intestine remains unclear. Our preliminary findings suggest that Imp1 loss may alter autophagy in intestinal epithelium during homeostasis and response to injury. Recent studies have linked aberrations in autophagy to Crohns disease. We therefore sought to determine if Imp1-mediated changes in autophagy may affect response to injury in the intestine epithelium and whether Imp1 expression is altered in Crohns disease patients. Methods:Mice with intestine-epithelial specific Imp1 deletion (VillinCre;Imp1fl/fl) were used to evaluate autophagy flux and response to irradiation or Heligmosomoides polygyrus infection via gene expression analyses, flow cytometry, and IHC/IF. Crypt enteroid assays were utilized to evaluate stem cell growth. Imp1 expression in Crohns disease patients was evaluated via qRT-PCR. Results:Imp1 expression is enriched in the crypt region of the small intestine, and VillinCre;Imp1fl/fl mice exhibit an increase in autophagy flux and enhanced expression of Paneth and stem cell markers in isolated crypts. Following challenge with irradiation or Heligmosomoides polygyrus infection, VillinCre;Imp1fl/fl mice exhibit robust crypt enteroid growth and improved clinical parameters consistent with Imp1 loss being protective in these contexts. Analysis of tissue biopsies from Crohns disease patients reveal a significant upregulation of Imp1 compared to unaffected patients, suggesting the possibility that overexpression of Imp1 may contribute to autophagy-related pathogenesis in Crohns disease. Conclusions:Our data demonstrate in 2 independent models that intestinal epithelial deletion of Imp1 promotes enhanced recovery from injury, possibly due to upregulation of stem cell gene expression and autophagy. Furthermore, our data reveal for the first time that Imp1 expression is increased in tissue from Crohns disease patients. Taken together, our findings may suggest a novel mechanism for IMP1 to promote pathogenesis of Crohns disease via negative regulation of autophagy.

380 Alcohol Induces ALDH2 As a Novel Cytoprotective Mechanism to Suppress Acetaldehyde-Derived DNA Adduct Formation in Esophageal Epithelial Cells

no statistically significant difference among different cell lines. We also treat CP-A Barretts cell line with acid (pH 6.5) for 24 hours and measured NOTCH signaling molecules by PCR array. We found that acid treatment significantly decreased NOTCH1 (2 fold decrease) and JAG1 (1.6 fold decrease), and increased NOTCH3 (1.7 fold increase) and NOTCH4 (3.9 fold increase) in CP-A Barretts cells. Acid treatment did not cause significant changes of DLL1, DLL2, DLL4, JAG2, NOTCH2 and NUMB. In addition, Western blot analysis showed that acid upregulated NOTCH3 and NOTCH4 protein expression. Acid-induced increase in cell proliferation in CP-A Barretts cells was significantly decreased by knockdown of NOTCH3 (from 285% control to 162% control, p<0.001) and NOTCH4 (from 236% control to 192% control, P<0.001) with their small interfering RNAs. We conclude that downregulation of NOTCH1 and overexpression of NOTCH3 may play an important role in the development of Barretts associated adenocarcinoma. It is possible that acid reflux present in BE patients may downregulate NOTCH1 and upregulate NOTCH3, increasing cell proliferation and thereby contributing to the progression from Barretts esophagus to esophageal adenocarcinoma. Supported by NIH NIDDK R01 DK080703.