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 remain unknown.
Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic three-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like1 (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 (ChIP) assays, quantitative reverse-transcription polymerase chain reactions (RT-PCR), Western blotting, immunofluorescence and immunohistochemistry.
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.
Notch signaling is important for esophageal epithelial homeostasis. In particular, the crosstalk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
NOTCH1; NOTCH3; esophageal epithelium; squamous differentiation
Genetic inactivation of Notch signaling in CD4−CD8− double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4+CD8+ double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein–tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRβ expression. DNMAML attenuated the pre-TCR–associated increase in cell size and CD27 expression. TCRβ or TCRαβ transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML− or DNMAML+ DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML+ DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the β-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J–MAML complex.
Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate (1) that is related to a propensity for invasion and metastasis (2). Here we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain of and loss of function experiments. Inhibition of EGFR signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (3D) culture can offer an important tool to discover novel biologic effectors in cancer.
tumor microenvironment; periostin; EGFR; p53
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. 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-like1 (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 (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D 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 squamous esophageal cancers.
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
Esophageal cancer (EC) ranks sixth in cancer death. To explore its genetic origins, we performed exomic sequencing on 11 adenocarcinomas (EAC) and 12 squamous cell carcinomas (ESCCs) from the United States. Interestingly, inactivating mutations of NOTCH1 were identified in 21% of ESCCs but not in EACs. There was a substantial disparity in the spectrum of mutations, with more indels in ESCCs, A:T>C:G transversions in EACs, and C:G>G:C transversions in ESCCs (p<0.0001). Notably, NOTCH1 mutations were more frequent in North American ESCCs (11 of 53 cases) than in ESCCs from China (1 of 48 cases). A parallel analysis found that most mutations in EACs were already present in matched Barrett’s esophagus (BE). These discoveries highlight key genetic differences between EAC and ESCC, American and Chinese ESCC, and suggest that NOTCH1 is a tumor suppressor gene in the esophagus. Finally, we provide a genetic basis for the evolution of EACs from BE.
Lymph node involvement and tumor-induced lymphangiogenesis appear as the earliest features of esophageal squamous cell carcinoma (ESCC), although the molecular regulatory mechanisms involved have remained unclear. Our aim was to investigate the contribution of NF-κB and Notch1 signaling to lymph node involvement and tumor-induced lymphangiogenesis in ESCC.
Material and methods
NF-κB and Notch1 expression in 60 tissue samples of ESCC were assessed by immunohistochemical staining. The correlations of NF-κB and Notch1 with lymph node involvement, lymphatic vessel density (LVD), podoplanin, and vascular endothelial growth factor-C (VEGF-C) were further evaluated to determine the association of NF-κB and Notch1 expression with tumor-induced lymphangiogenesis.
Chi-square tests revealed that NF-κB and Notch1 expression in ESCC tissues were significant associated with lymph node metastasis, LVD, podoplanin, and VEGF-C expression. Strong expression of NF-κB, but weak expression of Notch1, was observed in tumor tissues with lymph nodes involvement (P < 0.05 for both). The mean histoscores of LVD, podoplanin, and VEGF-C staining were higher in high-NF-κB-expressing tissue than in low-expressing tissue (P < 0.05 for each). In contrast, the mean histoscores of LVD and VEGF-C staining were lower in high-Notch1-expressing tissue than in low-expressing tissue (P < 0.05 for both). A multiple factors analysis of LVD and VEGF-C further demonstrated that LVD and VEGF-C status were significantly correlated with NF-κB and Notch1 expression in tumors. NF-κB and Notch1 expression were also significantly inversely correlated (P < 0.05).
These results suggest that different patterns of NF-κB and Notch1 signaling contribute to lymph nodes metastasis and tumor-induced lymphangiogenesis of ESCC, and reveal that up-regulation of NF-κB is associated with down-regulation of Notch1 in tumor tissue.
esophageal squamous cell carcinoma; Notch; NF-κB; angiogenesis; lymphangiogenesis
Background & Aims
Esophageal squamous cell carcinoma (ESCC) is known to be a highly angiogenic tumor. Here, we investigate the role of the stromal fibroblasts in the ESCC-induced angiogenic response using a novel 3D model.
A novel assay was developed where co-cultures of ESCC and esophageal fibroblasts induced HMVEC cell vascular network formation in a 3D collagen gel. Biochemical studies showed that the ESCC-induced activation of the fibroblasts was required to induce vascular network formation via a TGF-β and VEGF dependent pathway.
Conditioned media from a panel of 4 ESCC lines trans-differentiated normal esophageal fibroblasts into myofibroblasts via TGF-β signaling. The presence of fibroblasts was essential for efficient HMVEC network formation and the addition of ESCC cells to these cultures greatly enhanced the angiogenic process. The role of TGF-β in this process was demonstrated by the complete inhibition of network formation following TGF-β inhibitor treatment. Finally, we showed that ESCC-derived TGF-β regulates angiogenesis through the release of VEGF from the fibroblasts, and that the VEGF release was blocked following TGF-β inhibition.
This study demonstrates the essential role of fibroblasts in the ESCC angiogenic-induced response and suggests that the pharmacological targeting of the TGF-β signaling axis could be of therapeutic benefit in this deadly disease.
Aims. Nuclear translocation of β-catenin is common in many cancers including esophageal squamous cell carcinoma (ESCC). As a mediator of Wnt signaling pathway, nuclear β-catenin can activate many growth-related genes including CCN1, which in turn can induce β-catenin translocation. CCN1, a matricellular protein, signals through various integrin receptors in a cell-dependent manner to regulate cell adhesion, proliferation, and survival. Its elevation has been reported in ESCC as well as other esophageal abnormalities such as Barrett's esophagus. The aim of this study is to examine the relationship between CCN1 and β-catenin in ESCC. Methods and Results. The expression and correlation between CCN1 and β-catenin in ESCC tissue were examined through immunohistochemistry and further analyzed in both normal esophageal epithelial cells and ESCC cells through microarray, functional blocking and in situ protein ligation. We found that nuclear translocation of β-catenin in ESCC cells required high level of CCN1 as knockdown of CCN1 in ESCC cells reduced β-catenin expression and translocation. Furthermore, we found that integrin α11 was highly expressed in ESCC tumor tissue and functional blocking integrin α11 diminished CCN1-induced β-catenin elevation and translocation. Conclusions. Integrin α11 mediated the effect of CCN1 on β-catenin in esophageal epithelial cells.
The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4+-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.
esophageal cancer stem cell; esophageal squamous cell carcinoma; Nanog; Oct4; Sox2
The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression.
Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay.
In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC.
Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC.
The Notch1 gene plays an important role in mammalian cell fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signaling, we identified Epidermal Growth Factor Receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in the human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signaling in cancer cells counteracts the differentiation inducing effects of EGFR inhibitors, while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a novel role of EGFR signaling in negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches of cancer therapy.
Chemical Genetics; Squamous Cell Carcinoma; Keratinocytes; Notch; EGFR
FBXW7 is a tumor suppressor gene that induces the degradation of positive cell-cycle regulators such as c-Myc, cyclin E, c-Jun and Notch. The loss of FBXW7 promotes cell-cycle progression and cell proliferation. In the present study, we investigated the relationship between FBXW7 expression and the clinicopathological characteristics of patients with esophageal squamous cell carcinoma (ESCC). The expression of FBXW7 was quantified by real-time reverse transcription polymerase chain reaction in 43 primary ESCCs and their paired normal esophageal mucosa in patients who had not received preoperative therapy. FBXW7 expression levels were significantly correlated with the progression of the cancer and with local invasiveness. In muscle-invasive tumor cases (T2–4), lymphatic invasive tumor cases and stage II–IV cases, FBXW7 expression levels were significantly decreased (P=0.0315, P=0.0336 and P=0.0289, respectively). Decreased expression of FBXW7 was correlated with poor prognosis (P=0.0255). In conclusion, this study examined the relationship between FBXW7 expression and tumor progression in ESCC. We suggest that FBXW7 is a molecular prognostic marker and can be used to elucidate the mechanism of carcinogenesis.
esophageal cancer; prognosis; FBXW7
The aim of this study was to investigate the correlation between cyclin A expression and efficacy of paclitaxel-based chemotherapy in patients with esophageal squamous cell carcinoma (ESCC). The expression of cyclin A was examined in 48 newly diagnosed ESCC patients prior to treatment using the MaxVision immunohistochemistry method. The patients received four cycles of paclitaxel-based chemotherapy, the short-term treatment efficacy was evaluated and a 3-year follow-up was conducted. The response rate was greater in patients with positive cyclin A expression compared with those with negative expression (54.8 vs. 23.5%; χ2=4.373; P<0.05). Univariate and multivariate Cox analysis revealed that clinicopathological stage, degree of differentiation and expression of cyclin A were independent prognosis factors in patients with ESCC following paclitaxel-based chemotherapy. ESCC patients with positive cyclin A expression demonstrated an increased sensitivity to paclitaxel-based chemotherapy, suggesting that cyclin A may be used as a marker to predict the treatment efficacy of paclitaxel in patients with ESCC.
esophageal squamous cell carcinoma; cyclin A; paclitaxel; immunohistochemistry; chemotherapy
Gain-of-function experiments have demonstrated the potential of Notch signals to expand primitive hematopoietic progenitors, but whether Notch physiologically regulates hematopoietic stem cell (HSC) homeostasis in vivo is unclear. To answer this question, we evaluated the effect of global deficiencies of canonical Notch signaling in rigorous HSC assays. Hematopoietic progenitors expressing dominant negative Mastermind-like1 (DNMAML), a potent inhibitor of Notch-mediated transcriptional activation, achieved stable long-term reconstitution of irradiated hosts and showed a normal frequency of progenitor fractions enriched for long-term HSCs. Similar results were observed with cells lacking CSL/RBPJ, a DNA-binding factor that is required for canonical Notch signaling. Notch-deprived progenitors provided normal long-term reconstitution after secondary competitive transplantation. Furthermore, Notch target genes were expressed at low levels in primitive hematopoietic progenitors. Taken together, these results rule out an essential physiological role for cell-autonomous canonical Notch signals in HSC maintenance.
Imaging strategies that detect early-stage esophageal squamous cell carcinoma (ESCC) could improve clinical outcomes, combined with endoscopic approaches. Periostin is an integrin-binding protein that is important in the tumor microenvironment. We created a fluorescent-labeled antibody that recognizes periostin and binds specifically to ESCC xenograft tumors in mice. In L2-cre;p120ctnLoxP/LoxP mice, which develop squamous cell cancers that resemble human ESCC, we visualized the probe in preneoplastic and neoplastic esophageal lesions using near-infrared fluorescent imaging with upper gastrointestinal endoscopy. Periostin might be a biomarker of the esophageal tumor microenvironment that can be used to detect preneoplastic lesions.
mouse model; neoplasm; extracellular matrix; POSTN
The epidermal growth factor receptor (EGFR) signaling pathway regulates cell proliferation, differentiation, and survival, and is frequently dysregulated in esophageal and gastric cancers. Few studies have comprehensively examined the association between germline genetic variants in the EGFR pathway and risk of esophageal and gastric cancers. Based on a genome-wide association study in a Han Chinese population, we examined 3443 SNPs in 127 genes in the EGFR pathway for 1942 esophageal squamous cell carcinomas (ESCCs), 1758 gastric cancers (GCs), and 2111 controls. SNP-level analyses were conducted using logistic regression models. We applied the resampling-based adaptive rank truncated product approach to determine the gene- and pathway-level associations. The EGFR pathway was significantly associated with GC risk (P = 2.16×10−3). Gene-level analyses found 10 genes to be associated with GC, including FYN, MAPK8, MAP2K4, GNAI3, MAP2K1, TLN1, PRLR, PLCG2, RPS6KB2, and PIK3R3 (P<0.05). For ESCC, we did not observe a significant pathway-level association (P = 0.72), but gene-level analyses suggested associations between GNAI3, CHRNE, PAK4, WASL, and ITCH, and ESCC (P<0.05). Our data suggest an association between specific genes in the EGFR signaling pathway and risk of GC and ESCC. Further studies are warranted to validate these associations and to investigate underlying mechanisms.
Refluxogenic effects of smoking and alcohol abuse may be related to the risk of esophageal squamous cell carcinoma (ESCC). The present study attempts to clarify the effects of continuous taurocholic acid (TCA) exposure, which is neither mutagenic nor genotoxic, on ESCC progression.
A squamous carcinoma cell line (ESCC-DR) was established from a tumor induced in a rat model of gastroduodenal reflux. ESCC-DR cells were incubated with 2 mM TCA for ≥2 months. The effects of continuous TCA exposure were evaluated in vitro on cell morphology, growth, and invasion and in vivo on xenograft tumor growth in nude mice. Moreover, the mean level of secreted transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) proteins in cell culture supernatants and mRNA synthesis of TGF-β1 and VEGF-A of ESCC cells were measured. The angiogenic potential was further examined by a migration assay using human umbilical vein endothelial cells (HUVECs).
Continuous TCA exposure induced marked formation of filopodia in vitro. Expression levels of angiogenic factors were significantly higher in the cells treated with TCA than in control cells. Tumor xenografts derived from cells pre-exposed to TCA were larger and more vascularized than those derived from control cells. In addition, TCA exposure increased HUVEC migration.
Continuous TCA exposure enhanced ESCC progression due to reduced cell loss in vivo. Cell loss was inhibited by TCA-induced vascular endothelial cell migration, which was mediated by TGF-β1 and VEGF-A released from ESCC cells.
The aim of this study was to investigate the correlation between Wnt1/β-catenin expression and the clinicopathologic features and prognosis of patients with esophageal squamous cell carcinoma (ESCC). The mRNA and protein expression levels of Wnt1/β-catenin genes in 70 ESCC and 15 adjacent noncancerous paraffin-embedded samples were determined by real-time quantitative polymerase chain reaction and immunohistochemical staining. The mRNA expression level of Wnt1/β-catenin in ESCC was significantly higher than that in the adjacent noncancerous tissues (1.9934 ± 1.9888 vs. 0.8863 ± 0.665, p = 0.0184; 0.2854 ± 0.1298 vs. 0.0128 ± 0.0158, p = 0.0000, respectively), and the overexpression of Wnt1/β-catenin mRNA was aggressively associated with lymph node metastasis and advanced pathological stage (p < 0.0001). The protein expression level of Wnt1/β-catenin was also significantly higher than that in the adjacent noncancerous tissues (0.3830 ± 0.0947 vs. 0.2721 ± 0.1474, p = 0.0002; 0.2835 ± 0.0844 vs. 0.2352 ± 0.0670, p = 0.0210, respectively); however, the overexpression was not associated with clinicopathologic characteristics. Meanwhile, the protein expression level of Wnt1 had no relevance with that of β-catenin. The overexpression of Wnt1/β-catenin might be an important molecular marker to predict the clinicopathologic stage and prognosis of ESCC, and the level of Wnt1/β-catenin mRNA was conversely correlated with lymph node metastasis and advanced pathological stage. The overexpression of Wnt1/β-catenin mRNA should also predict poor prognosis of ESCC; however, it might not be an independent prognostic factor.
Esophageal Squamous Cell Carcinoma (ESCC) is a major subtype of esophageal cancer causing significant morbility and mortality in Asia. Mechanism of initiation and progression of this disease is unclear. Tumor initiating cells (TICs) are the subpopulation of cells which have the ability to self-renew, as well as, to drive initiation and progression of cancer. Increasing evidence has shown that TICs exist in a variety of tumors. However, the identification and characterization of TICs in esophageal carcinoma has remained elusive.
to identify TICs in ESCC, ESCC cell lines including two primary cells were used for screening suitable surface marker. Then colony formation assay, drug resistant assay and tumorigenicity assay in immune deficient mice were used to characterize TICs in ESCC. We found that just the CD44 expression correlated with tumorigenicity in ESCC cell lines. And then induced differentiation of ESCC cells by all-trans retinoic acid treatment led to decreased expression of CD44. The FACS isolated cell subpopulations with high CD44 expression showed increased colony formation and drug resistance in vitro, as well as significantly enhanced tumorigenicity in NOD/SICD mice, as compared to the low expressing CD44 ESCC cells.
our study has discovered a novel TIC surface marker, CD44, which can be utilized to enrich efficiently the TICs in ESCC. These findings will be useful for further studies of these cells and exploring therapeutic approaches.
Slug, a regulator of epithelial mesenchymal transition, was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. This study aimed to determine the clinical significance of Slug overexpression in ESCC and determine its correlation with clinicopathological parameters and disease prognosis for ESCC patients.
Immunohistochemical analysis of Slug expression was carried out in archived tissue sections from 91 ESCCs, 61 dysplastic and 47 histologically normal esophageal tissues. Slug immunopositivity in epithelial cells was correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients.
Increased expression of Slug was observed in esophageal dysplasia [cytoplasmic, 24/61 (39.3%) cases, p = 0.001, odd’s ratio (OR) = 4.7; nuclear, 11/61 (18%) cases, p < 0.001, OR = 1.36] in comparison with normal esophageal tissues. The Slug expression was further increased in ESCCs [cytoplasmic, 64/91 (70.3%) p < 0.001, OR = 10.0; nuclear, 27/91 (29.7%) p < 0.001, OR = 1.42]. Kaplan Meier survival analysis showed significant association of nuclear Slug accumulation with reduced disease free survival of ESCC patients (median disease free survival (DFS) = 6 months, as compared to those that did not show overexpression, DFS = 18 months; p = 0.006). In multivariate Cox regression analysis nuclear Slug expression [p= 0.005, Hazard’s ratio (HR) = 2.269, 95% CI = 1.289 - 3.996] emerged as the most significant independent predictor of poor prognosis for ESCC patients.
Alterations in Slug expression occur in early stages of development of ESCC and are sustained during disease progression. Slug may serve as a diagnostic biomarker and as a predictor of poor disease prognosis to identify ESCC patients that are likely to show recurrence of the disease.
Epidermal growth factor receptor (EGFR) is suggested to predict the radiosensitivity and/or prognosis of human esophageal squamous cell carcinoma (ESCC). The objective of this study was to investigate the efficacy of Nimotuzumab (an anti-EGFR monoclonal antibody) on ESCC radiotherapy (RT) and underlying mechanisms.
Nimotuzumab was administrated to 2 ESCC cell lines KYSE30 and TE-1 treated with RT. Cell growth, colony formation and apoptosis were used to measure anti-proliferation effects. The method of RNA interference was used to investigate the role of insulin-like growth factor binding protein-3 (IGFBP-3) in ESCC cells radiosensitivity treated with Nimotuzumab. In vivo effect of Nimotuzumab on ESCC radiotherapy was done using a mouse xenograft model.
Nimotuzumab enhanced radiation response of KYSE30 cells (with high EGFR expression) in vitro, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. Mechanism study revealed that Nimotuzumab inhibited phosphorylated EGFR (p-EGFR) induced by EGF in KYSE30 cells. In addition, knockdown of IGFBP-3 by short hairpin RNA significantly reduced KYSE30 cells radiosensitivity (P<0.05), and even after the administration of Nimotuzumab, the RT response of IGFBP-3 silenced KYSE30 cells was not enhanced (P>0.05). In KYSE30 cell xenografts, Nimotuzumab combined with radiation led to significant tumor growth delay, compared with that of radiation alone (P=0.029), and also with IGFBP-3 up-regulation in tumor tissue.
Nimotuzumab could enhance the RT effect of ESCC cells with a functional active EGFR pathway. In particular, the increased ESCC radiosensitivity by Nimotuzumab might be dependent on the up-regulation of IGFBP-3 through EGFR-dependent pathway.
EGFR; Esophageal squamous carcinoma cell; IGFBP-3; Nimotuzumab; Radiosensitivity
Esophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.
Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.
Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.
These results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.
Continuous exposure to various environmental carcinogens and genetic polymorphisms of xenobiotic-metabolizing enzymes (XME) are associated with many types of human cancers, including esophageal squamous cell carcinoma (ESCC). Huaian, China, is one of the endemic regions of ESCC, but fewer studies have been done in characterizing the risk factors of ESCC in this area. The aims of this study is to evaluate the etiological roles of demographic parameters, environmental and food-borne carcinogens exposure, and XME polymorphisms in formation of ESCC, and to investigate possible gene-gene and gene-environment interactions associated with ESCC in Huaian, China.
A population based case-control study was conducted in 107 ESCC newly diagnosed cases and 107 residency- age-, and sex-matched controls in 5 townships of Huaian. In addition to regular epidemiological and food frequency questionnaire analyses, genetic polymorphisms of phase I enzymes CYP1A1, CYP1B1, CYP2A6, and CYP2E1, and phase II enzymes GSTM1, GSTT1, GSTP1, and microsomal epoxide hydrolase (EPHX) were assessed from genomic DNA using PCR based techniques.
Consuming acrid food, fatty meat, moldy food, salted and pickled vegetables, eating fast, introverted personality, passive smoking, a family history of cancer, esophageal lesion, and infection with Helicobacter pylori were significant risk factors for ESCC (P < 0.05). Regular clean up of food storage utensils, green tea consumption, and alcohol abstinence were protective factors for ESCC (P < 0.01). The frequency of the GSTT1 null genotype was higher in cases (59.4%) compared to controls (47.2%) with an odds ratio (OR) of 1.68 and 95% confidence interval (CI) from 0.96 to 2.97 (P = 0.07), especially in males (OR = 2.78; 95% CI = 1.22–6.25; P = 0.01). No associations were found between polymorphisms of CYP1A1, CYP1B1, CYP2A6, CYP2E1, GSTM1, GSTP1, and EPHX and ESCC (P > 0.05).
Our results demonstrated that dietary and environmental exposures, some demographic parameters and genetic polymorphism of GSTT1 may play important roles in the development of ESCC in Huaian area, China.
Overexpression of Cyclooxygenase-2 (COX-2) was observed in many types of cancers, including esophageal squamous cell carcinoma (ESCC). One functional SNP, COX-2 -1195G/A, has been reported to mediate susceptibility of ESCC in Chinese populations. In our previous study, the presence of Helicobacter pylori (H. pylori) was found to play a protective role in development of ESCC. The interaction of COX-2 and H. pylori in gastric cancer was well investigated. However, literature on their interaction in ESCC risk is scarce. The purpose of this study was to evaluate the association and interaction between COX-2 single nucleotide polymorphism (SNP), H. pylori infection and the risk of developing ESCC.
One hundred and eighty patients with ESCC and 194 controls were enrolled in this study. Personal data regarding related risk factors, including alcohol consumption, smoking habits and betel quid chewing, were collected via questionnaire. Genotypes of the COX-2 -1195 polymorphism were determined by PCR-based restriction fragment length polymorphism. H. pylori seropositivity was defined by immunochromatographic screening test. Data was analyzed by chi-squared tests and polytomous logistics regression.
In analysis adjusting for the covariates and confounders, H. pylori seropositivity was found to be inversely association with the ESCC development (adjusted OR: 0.5, 95% CI: 0.3 – 0.9). COX-2 -1195 AA homozygous was associated with an increased risk of contracting ESCC in comparison with the non-AA group, especially among patients with H. pylori seronegative (adjusted OR ratio: 2.9, 95% CI: 1.2 – 7.3). The effect was strengthened among patients with lower third ESCC (adjusted OR ratio: 6.9, 95% CI 2.1 – 22.5). Besides, H. pylori seropositivity conveyed a notably inverse effect among patients with COX-2 AA polymorphism (AOR ratio: 0.3, 95% CI: 0.1 – 0.9), and the effect was observed to be enhanced for the lower third ESCC patients (AOR ratio: 0.09, 95% CI: 0.02 – 0.47, p for multiplicative interaction 0.008)
H. pylori seropositivity is inversely associated with the risk of ESCC in Taiwan, and COX-2 -1195 polymorphism plays a role in modifying the influence between H. pylori and ESCC, especially in lower third esophagus.
Hedgehog (Hh) signaling is frequently activated in human cancer, including
esophageal cancer. Most esophageal cancers are diagnosed in the advanced stages,
therefore, identifying the very alterations that drive esophageal carcinogenesis
may help designing novel strategies to diagnose and treat the disease. Analysis
of Hh signaling in precancerous lesions is a critical first step in determining
the significance of this pathway for carcinogenesis. Here we report our data on
Hh target gene expression in 174 human esophageal specimens [28 esophageal
adenocarcinomas (EAC), 19 Barrett’s esophagus, 103 cases of esophageal
squamous cell carcinoma (ESCC), and 24 of squamous dysplastic lesions], and in
two rat models of esophageal cancer. We found that 96% of human EAC express Hh
target genes. We showed that PTCH1 expression is the most reliable biomarker. In
contrast to EAC, only 38% of ESCC express Hh target genes. We found activation
of Hh signaling in precancerous lesions of ESCCs and EACs in different degrees
(21% and 58% respectively). Expression of Hh target genes is frequently detected
in severe squamous dysplasia/ carcinoma in situ (p=0.04) and
Barrett’s esophagus (p=0.01). Unlike EAC, sonic hedgehog (Shh) expression
was rare in ESCCs. Consistent with the human specimen data, we found a high
percentage of Hh signaling activation in precancerous lesions in rat models.
These data indicate that Hh signaling activation is an early molecular event in
the development of esophageal cancer, particularly EAC.
Esophageal adenocarcinoma (EAC); esophageal squamous cell carcinoma (ESCC); hedgehog (Hh); patched-1 (PTCH1 for humans and Ptch1 for animals); Gli2; sFRP-1; human homologue of hedgehog-interaction protein (HHIP); rat model; Barrett’s esophagus (BE)