NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.

BACKGROUND & AIMS: The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation, and proliferation in the hematopoietic system, the intestine, the pancreas, and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology are unknown. METHODS: Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic 3-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like 1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation assays, quantitative reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, and immunohistochemistry. RESULTS: NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia. CONCLUSIONS: Notch signaling is important for esophageal epithelial homeostasis. In particular, the cross talk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.

Insulin-like growth factor-binding protein-3 promotes transforming growth factor-{beta}1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells.

Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-beta1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2-hTERT-EGFR-p53(R175H). In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-beta target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-beta1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-beta1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3(I56G/L80G/L81G), a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-beta1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3(I56G/L80G/L81G) also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-beta1-mediated EMT and cell invasion.

A mammalian melanopsin in the retina of a fresh water turtle, the red-eared slider (Trachemys scripta elegans).

A mammalian-like melanopsin (Opn4m) has been found in all major vertebrate classes except reptile. Since the pupillary light reflex (PLR) of the fresh water turtle takes between 5 and 10 min to achieve maximum constriction, and since photosensitive retinal ganglion cells (ipRGCs) in mammals use Opn4m to control their slow sustained pupil responses, we hypothesized that a Opn4m homolog exists in the retina of the turtle. To identify its presence, retinal tissue was dissected from seven turtles, and total RNA extracted. Reverse transcriptase-polymerase chain reactions (RT-PCRs) were carried out to amplify gene sequences using primers targeting the highly conserved core region of Opn4m, and PCR products were analyzed by gel electrophoresis and sequenced. Sequences derived from a 1004-bp PCR product were compared to those stored in GenBank by the basic local alignment search tool (BLAST) algorithm and returned significant matches to several Opn4ms from other vertebrates including chicken. Quantitative real-time PCR (qPCR) was also carried out to compare expression levels of Opn4m in different tissues. The normalized expression level of Opn4m in the retina was higher in comparison to other tissue types: iris, liver, lung, and skeletal muscle. The results suggest that Opn4m exists in the retina of the turtle and provides a possible explanation for the presence of a slow PLR. The turtle is likely to be a useful model for further understanding the photoreceptive mechanisms in the retina which control the dynamics of the PLR.

A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors.

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-ß-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-ß-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.

Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors.

Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.

Trainees do not negatively impact the institutional learning curve for robotic prostatectomy as characterized by operative time, estimated blood loss, and positive surgical margin rate.

OBJECTIVES: We evaluated the learning curves and perioperative outcomes of an experienced laparoscopic surgeon and his trainees to assess our structured teaching program. METHODS: We retrieved 383 patients undergoing robot-assisted laparoscopic prostatectomy (RALP) from our database. Trainees completed a structured teaching program and were categorized as early (days 0 to 232), mid (days 566 to 797), and late (days 825 to 1218) according to the time period in which they were working with the mentor. We compared operative times, estimated blood loss (EBL), and positive surgical margin (PSM) rates between the trainees and the mentor (Mann-Whitney and Chi-square test). Association of EBL, body mass index (BMI), and prostate weight with operative time was evaluated in multivariate linear regression analysis. RESULTS: Median operative times of the early, mid, and late trainees (258, 220, and 200 minutes) significantly decreased and were similar to the corresponding senior surgeon's (254, 242, and 180 minutes). Operative times decreased with lower BMI, EBL, and prostate weight (P = 0.006, P <0.001, and P <0.001, respectively). Overall, EBL (150 mL vs. 150 mL, P = 0.215) and PSM rates (20% vs. 18.6%, P = 0.741) did not differ between the mentor and the trainees. CONCLUSIONS: A structured teaching program for RALP is effective and trainees are able to adopt the increased efficiency and skills of their mentor. Lower BMI, EBL, and prostate weight were associated with shorter operative times. Trainees performing the procedure did not negatively affect EBL and positive surgical margin rate.