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:In multiple myeloma (MM), communication via Notch signaling in the tumor niche stimulates tumor progression and bone destruction. We previously showed that osteocytes activate Notch, increase Notch3 expression, and stimulate proliferation in MM cells. We show here that Notch3 inhibition in MM cells reduced MM proliferation, decreased Rankl expression, and abrogated the ability of MM cells to promote osteoclastogenesis. Further, Notch3 inhibition in MM cells partially prevented the Notch activation and increased proliferation induced by osteocytes, demonstrating that Notch3 mediates MM-osteocyte communication. Consistently, pro-proliferative and pro-osteoclastogenic pathways were upregulated in CD138+ cells from newly diagnosed MM patients with high vs. low NOTCH3 expression. These results show that NOTCH3 signaling in MM cells stimulates proliferation and increases their osteoclastogenic potential. In contrast, Notch2 inhibition did not alter MM cell proliferation or communication with osteocytes. Lastly, mice injected with Notch3 knock-down MM cells had a 50% decrease in tumor burden and a 50% reduction in osteolytic lesions than mice bearing control MM cells. Together, these findings identify Notch3 as a mediator of cell communication among MM cells and between MM cells and osteocytes in the MM tumor niche and warrant future studies to exploit Notch3 as a therapeutic target to treat MM.
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Project description:BackgroundEpithelial-mesenchymal transition (EMT) and cell stemness are implicated in the initiation and progression of oral squamous cell carcinoma (OSCC). Revealing the intrinsic regulatory mechanism may provide effective therapeutic targets for OSCC.ResultsIn this study, we found that Forkhead box D1 (FOXD1) was upregulated in OSCC compared with normal samples. Patients with a higher FOXD1 expression had a poorer overall survival and disease-free survival. Immunohistochemical staining results showed that FOXD1 expression was related to the clinical stage and relapse status of OSCC patients. When FOXD1 expression was knocked down in CAL27 and SCC25 cells, the migration, invasion, colony formation, sphere formation, and proliferation abilities decreased. Moreover, EMT and stemness-related markers changed remarkably, which indicated that the EMT process and cell stemness were inhibited. Conversely, overexpression of FOXD1 promoted EMT and cell stemness. Further study demonstrated that FOXD1 could bind to the promoter region and activate the transcription of SNAI2. In turn, the elevated SNAI2 affected EMT and cell stemness. An in vivo study showed that FOXD1-overexpressing CAL27 cells possessed a stronger tumorigenic ability.ConclusionsOur findings revealed a novel mechanism in regulating EMT and cell stemness and proposed FOXD1 as a potential marker for the diagnosis and treatment of OSCC.
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