Wen Wen Xu

Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance

Purpose: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. Experimental Design: In vitro and in vivo functional assays were performed to study the effects of Id1–E2F1–IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. Results: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. Conclusions: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1–E2F1–IGF2 regulatory axis has important implications for cancer prognosis and treatment. Clin Cancer Res; 22(5); 1243–55. ©2015 AACR.

Significance of PI3K/AKT signaling pathway in metastasis of esophageal squamous cell carcinoma and its potential as a target for anti-metastasis therapy

Metastasis is the most lethal hallmark of esophageal squamous cell carcinoma (ESCC). The aim of the study is to identify key signaling pathways that control metastasis in ESCC. Highly invasive ESCC sublines (designated I3 cells) were established through three rounds of selection of cancer cells invading through matrigel-coated chambers. Gene expression profile of one of the I3 sublines was compared with that of its parental cell line using cDNA microarray analysis. Gene ontology and pathway analyses of the differentially expressed genes (both upregulated and downregulated) indicated that genes associated with cellular movement and the AKT pathway were associated with increased cancer cell invasiveness. Western blot analysis confirmed increased phosphorylated AKT (p-AKT), N-cadherin and decreased E-cadherin expression in the I3 cells. Immunohistochemistry was used to evaluate the clinical significance of p-AKT expression in ESCC, and the results showed higher p-AKT nuclear expression in lymph node metastases when compared with primary carcinoma. Inactivation of the PI3K/AKT pathway with specific inhibitors, or with PTEN overexpression, resulted in reversed cadherin switching and inhibited cancer cell motility. Inhibition of the pathway by treatment with wortmannin markedly suppressed experimental metastasis in nude mice. Our data demonstrated the importance of the PI3K/AKT signaling pathway in ESCC metastasis and support PI3K/AKT as a valid therapeutic target in treatment of metastatic ESCC.

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3′ untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression.

Role of AMPK signaling in mediating the anticancer effects of silibinin in esophageal squamous cell carcinoma

Objective: Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK) may suppress cancer growth. Identification of novel AMPK activators is therefore crucial to exploit AMPK as a potential target for cancer prevention and treatment. Research design and methods: We determined the expression status and role of AMPK in esophageal squamous cell carcinoma (ESCC) and investigated whether silibinin, a nontoxic natural product, could activate AMPK to inhibit ESCC development. Results: Our results from 49 pairs of human ESCC and normal tissues showed that AMPK was constitutively inactive in the majority (69.4%) of ESCC. We found that silibinin induced apoptosis, and inhibited ESCC cell proliferation in vitro and tumorigenicity in vivo without any adverse effects. Silibinin also markedly suppressed the invasive potential of ESCC cells in vitro and their ability to form lung metastasis in nude mice. The anticancer effects of silibinin were abrogated by the presence of compound C or shRNA against AMPK. More importantly, silibinin enhanced the sensitivity of ESCC cells and tumors to the chemotherapeutic drugs, 5-fluorouracil and cisplatin. Conclusions: This preclinical study supports that AMPK is a valid therapeutic target and suggests that silibinin may be a potentially useful therapeutic agent and chemosensitizer for esophageal cancer.

Comparative proteomics analysis identifies Cdc42-Cdc42BPA signaling as prognostic biomarker and therapeutic target for colon cancer invasion

Metastasis is one of the major causes of treatment failure in the patients with colon cancer. The aim of our study is to find key proteins and pathways that drive invasion and metastasis in colon cancer. Eight rounds of selection of cancer cells invading through matrigel-coated chamber were performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture technology was used to identify the differently expressed proteins, and the proteomics data were analyzed by ingenuity pathway analysis. PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes. HCT116-I8 and RKO-I8 cells showed significantly stronger invasive potential as well as decreased E-cadherin and increased vimentin expressions compared with parental cells. The differently expressed proteins in I8 cells compared with parental cells were identified. Bioinformatics analysis of proteomics data suggested that Cdc42BPA protein and Cdc42 signaling pathway are important for colon cancer invasion, which was confirmed by experimental data showing upregulation of Cdc42BPA and higher expression of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon cancer cell invasion and reversed epithelial mesenchymal transition process. Furthermore, compared with adjacent normal tissues, Cdc42BPA expression was significantly higher in colon cancer tissues and further upregulated in metastatic tumors in lymph nodes. More importantly, Cdc42BPA expression was correlated with metastasis and poor survival of the patients with colon cancer. This study provides the first evidence that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion, and Cdc42BPA has potential implications for colon cancer prognosis and treatment.

Synephrine hydrochloride suppresses esophageal cancer tumor growth and metastatic potential through inhibition of Galectin-3-AKT/ERK signaling

A library consisting of 429 food-source compounds was used to screen the natural products with anticancer properties in esophageal squamous cell carcinoma (ESCC). We demonstrated for the first time that synephrine, an active compound isolated from leaves of citrus trees, markedly suppressed cell proliferation (inhibition rate with 20 μM synephrine at day 5:71.1 ± 5.8% and 75.7 ± 6.2% for KYSE30 and KYSE270, respectively) and colony formation (inhibition rate with 10 μM synephrine: 86.5 ± 5.9% and 82.3 ± 4.5% for KYSE30 and KYSE270, respectively), as well as migration (inhibition rate with 10 μM synephrine: 76.9 ± 4.4% and 62.2 ± 5.8% for KYSE30 and KYSE270, respectively) and invasion abilities (inhibition rate with 10 μM synephrine: 73.3 ± 7.5% and 75.3 ± 3.4% for KYSE30 and KYSE270, respectively) of ESCC cells in a dose-dependent manner, without significant toxic effect on normal esophageal epithelial cells. Mechanistically, quantitative proteomics and bioinformatics analyses were performed to explore the synephrine-regulated proteins. Western blot and qRT-PCR data indicated that synephrine may downregulate Galectin-3 to inactivate AKT and ERK pathways. In addition, we found that the sensitivity of ESCC to fluorouracil (5-FU) could be enhanced by synephrine. Furthermore, in vivo experiments showed that synephrine had significant antitumor effect on ESCC tumor xenografts in nude mice (inhibition rate with 20 mg/kg synephrine is 61.3 ± 20.5%) without observed side effects on the animals. Taken together, synephrine, a food-source natural product, may be a potential therapeutic strategy in ESCC.

Identification of miR-29c/FBXO31 as a key regulatory mechanism in esophageal cancer chemoresistance

C patients treated with chemotherapy may show impaired cognition (Chemofog) long-term after the treatment completion. Recently, targeted therapies have been developed and have been also associated with the appearance of leukoencephalopathy and major asthenia in cancer patients. Our previous work has identified a direct link between chemotherapy and impaired long-term behavioral flexibility associated with a decreased proliferation within the hippocampus and no cognitive dysfunctions after the cancer treatment everolimus in mice. In this study, we evaluated the impact of the systemic administration of an anti-VEGF antibody on cognitive function, hippocampal vascularization, cerebral metabolic activity and hippocampal synaptic activity in mice as well as on proliferation of neural stem and endothelial cells in vitro. Antibodies against the mouse VEGF (B20-4.1.1, MTA Genentech) and human VEGF (bevacizumab, Genentech, MTA, Roche) were administered to adult C57/Bl6 mice (1.5 mg/kg) every 4 days for 24 days. Note that B20-4.1.1 treatment caused a slowing of weight gain. During/or after treatment, emotional reactivity, spontaneous activity, learning and spatial memory, behavioral flexibility and object recognition memory were assessed. The selective cognitive impairments observed in B20-treated mice (spatial learning in the Morris water maze and memory consolidation in the object recognition test), were predictive of hippocampal dysfunctions. In addition, we detected modifications of the cytochrome oxidase activity in CA1-CA3 hippocampal area and markers involved in long term potentiation. However, proliferation of neural hippocampal precursors (BrdU labeling) and vascular density (Collagen IV) in vivo as well as neurosphere growth and Bend.3 endothelial cell proliferation in vitro were unaltered. Together, these data indicate that the inhibition of endogenous systemic VEGF levels does not drastically change the plasticity of the hippocampal vascular niche, but selectively alters the spatial learning dependent on the long-term potentiation in the hippocampus.

The potential of targeted antiangiogenesis therapies in the treatment of esophageal cancer

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Gastrointestinal Cancer: Targets and Therapy 2015:5 79–88 Gastrointestinal Cancer: Targets and Therapy Dovepress

Role of miR-29c in modulating the paracrine crosstalk between esophageal cancer cells and stromal fibroblasts in the tumor microenvironment.

C patients treated with chemotherapy may show impaired cognition (Chemofog) long-term after the treatment completion. Recently, targeted therapies have been developed and have been also associated with the appearance of leukoencephalopathy and major asthenia in cancer patients. Our previous work has identified a direct link between chemotherapy and impaired long-term behavioral flexibility associated with a decreased proliferation within the hippocampus and no cognitive dysfunctions after the cancer treatment everolimus in mice. In this study, we evaluated the impact of the systemic administration of an anti-VEGF antibody on cognitive function, hippocampal vascularization, cerebral metabolic activity and hippocampal synaptic activity in mice as well as on proliferation of neural stem and endothelial cells in vitro. Antibodies against the mouse VEGF (B20-4.1.1, MTA Genentech) and human VEGF (bevacizumab, Genentech, MTA, Roche) were administered to adult C57/Bl6 mice (1.5 mg/kg) every 4 days for 24 days. Note that B20-4.1.1 treatment caused a slowing of weight gain. During/or after treatment, emotional reactivity, spontaneous activity, learning and spatial memory, behavioral flexibility and object recognition memory were assessed. The selective cognitive impairments observed in B20-treated mice (spatial learning in the Morris water maze and memory consolidation in the object recognition test), were predictive of hippocampal dysfunctions. In addition, we detected modifications of the cytochrome oxidase activity in CA1-CA3 hippocampal area and markers involved in long term potentiation. However, proliferation of neural hippocampal precursors (BrdU labeling) and vascular density (Collagen IV) in vivo as well as neurosphere growth and Bend.3 endothelial cell proliferation in vitro were unaltered. Together, these data indicate that the inhibition of endogenous systemic VEGF levels does not drastically change the plasticity of the hippocampal vascular niche, but selectively alters the spatial learning dependent on the long-term potentiation in the hippocampus.