Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma.
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ABSTRACT:
SUBMITTER: Natsuizaka M
PROVIDER: S-EPMC5700926 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The highest incidence of esophageal squamous cell carcinoma (ESCC) occurs in China. Cancer stem cells play key roles for tumor progression. Gene amplified in squamous cell carcinoma 1 (GASC1) is essential to maintain self-renewal and differentiation potential of embryonic stem cells. This study aimed to reveal the effect and mechanism of GASC1 on ESCC stemness. The biological function of GASC1 in ESCC was evaluated both in vitro and in vivo. ChIP assay was performed to determine the molecular mechanism of GASC1 in epigenetic regulation of NOTCH1. We found that GASC1 expression was increased in poor differentiated ESCC cells and tissues. ESCC patients with a high level of GASC1 presented a significantly worse survival rate. GASC1 expression in purified ALDH+ ESCC cells was significantly higher than that in ALDH- cells. The stemness of ESCC was dramatically decreased after GASC1 blockade. Furthermore, blockade of GASC1 decreased NOTCH1 expression via increase of NOTCH1 promoter H3K9me2 and H3K9me3. Moreover, the impaired stemness after blockade of GASC1 could be reversed after transfection of NOTCH1 overexpression lentiviral vector. GASC1 promoted stemness in ESCC cells via NOTCH1 promoter demethylation. Therefore, GASC1/NOTCH1 signaling might be a potential therapeutic target for the treatment of ESCC patients.
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Project description:The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-j?, 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.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 ?-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.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 cross talk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
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Project description:Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.
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Project description:Frizzled (FZD) proteins are receptors for secreted WNT proteins and play a critical role in the malignant progression of various cancers. However, the role of human FZD family members in esophageal squamous cell carcinoma (ESCC) was rarely investigated. In this study, we found that the FZD7 gene was the most commonly up-regulated FZD member in ESCC cell lines compared with other FZDs. TMA studies further validated that FZD7 protein was up-regulated in 165 of 252 (65.5%) informative ESCC patients and significantly correlated with poor overall survival (P=0.001). Additionally, multivariate Cox regression analysis showed that FZD7 overexpression was an independent prognostic factor for ESCC patients. Ectopic expression of FZD7 could promote ESCC cell metastasis both in vitro and in vivo. Under WNT3A stimulation, FZD7 was able to induce the nuclear translocation of ?-catenin and activate the downstream targets of WNT/?-catenin signaling, as well as promote epithelial-mesenchymal transition (EMT) potential in ESCC cells. Our study demonstrated for the first time that FZD7 contributes to the malignant progression of ESCC and represents a novel prognostic marker and a potential therapeutic target for ESCC patients.
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Project description:Transforming growth factor β1 (TGF-β1) plays an important role in tumor initiation and development by inducing epithelial-mesenchymal Transition (EMT). Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a long noncoding RNA (lncRNA) that contributes to the invasion and metastasis of tumors, including esophageal squamous cell carcinoma (ESCC). The aim of the present study was to explore the underlying mechanisms implicated in EMT and to clarify whether TGF-β1 regulates MALAT1 expression, thereby promoting the invasion of ESCC. Expression of TGF-β1, MALAT1 and EMT-related markers, including E-cadherin and Vimentin, was detected in clinical samples of Kazakh's ESCC. The role of TGF-β1 in the regulation of MALAT1 in ESCC invasion was evaluated at the ESCC cell line level. High TGF-β1 expression was significantly associated with poor survival among patients with Kazakh's ESCC. Additionally, the expression of Vimentin was upregulated, and the expression of E-cadherin was downregulated and varied. The expression of MALAT1 positively correlated with the expression of TGF-β1 both in vivo and in vitro. Furthermore, knockdown of MALAT1 inhibited TGF-β1-induced EMT. Our data indicate that MALAT1 is heavily involved in EMT induced by TGF-β1. MALAT1 may be a therapeutic target in the suppression of metastasis and invasion of ESCC.
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Project description:BackgroundCancer-associated fibroblasts (CAFs), an important component of the tumor microenvironment (TME), play crucial roles in tumor stemness. It has been shown in various cancer studies that stanniocalcin-1 (STC1) is secreted by CAFs, however, its function in HCC is still not clear.MethodsThe serum concentration and intracellular expression level of STC1 were quantified by ELISA and western blotting, respectively. The role of CAF-derived STC1 in HCC stemness was investigated by sphere formation, sorafenib resistance, colony formation, and transwell migration and invasion assays in vitro and in an orthotopic liver xenograft model in vivo. An HCC tissue microarray containing 72 samples was used to evaluate the expression of STC1 and Notch1 in HCC tissues. Coimmunoprecipitation (CoIP) and dual-luciferase reporter assays were performed to further explore the underlying mechanisms. ELISAs were used to measure the serum concentration of STC1 in HCC patients.ResultsWe demonstrated that CAFs were the main source of STC1 in HCC and that CAF-derived STC1 promoted HCC stemness through activation of the Notch signaling pathway. In HCC patients, the expression of STC1 was positively correlated with Notch1 expression and poor prognosis. The co-IP assay showed that STC1 directly bound to Notch1 receptors to activate the Notch signaling pathway, thereby promoting the stemness of HCC cells. Our data further demonstrated that STC1 was a direct transcriptional target of CSL in HCC cells. Furthermore, ELISA revealed that the serum STC1 concentration was higher in patients with advanced liver cancer than in patients with early liver cancer.ConclusionsCAF-derived STC1 promoted HCC stemness via the Notch1 signaling pathway. STC1 might serve as a potential biomarker for the prognostic assessment of HCC, and the stromal-tumor amplifying STC1-Notch1 feedforward signal could constitute an effective therapeutic target for HCC patients.
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