Fang Sui

Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

The biological function of E26 transformation-specific (ETS) transcription factor EHF/ESE-3 in human cancers remains largely unknown, particularly gastric cancer. The aim of this study was to explore the role of EHF in tumorigenesis and its potential as a therapeutic target in gastric cancer. By using quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays, we investigated the expression and copy number of EHF in a cohort of gastric cancers and control subjects. Specific EHF siRNAs was used to determine the biologic impacts and mechanisms of altered EHF expression in vitro and in vivo. Dual-luciferase reporter, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) assays were performed to identify its downstream targets. Our results demonstrated that EHF was significantly upregulated and frequently amplified in gastric cancer tissues as compared with control subjects. Moreover, EHF amplification was positively correlated with its overexpression and significantly associated with poor clinical outcomes of gastric cancer patients. We also found that EHF knockdown notably inhibited gastric cancer cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice and induced cell cycle arrest and apoptosis. Importantly, we identified EHF as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. Collectively, our findings suggest that EHF is a novel functional oncogene in gastric cancer by regulating the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and may represent a potential prognostic marker and therapeutic target for this cancer.

PAX3 is a novel tumor suppressor by regulating the activities of major signaling pathways and transcription factor FOXO3a in thyroid cancer

Paired box 3 (PAX3) is expressed early during embryonic development in spatially restricted domains in the nervous system and in some mesodermally-derived structure. In recent years, it is found to be overexpressed in different types of cancer tissues and cell lines including glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas and gastric cancers, suggesting that it may function as an oncogene in these cancers. However, its role in thyroid cancer remains totally unclear. The aim of this study was to explore the functions and related molecular mechanism of PAX3 in thyroid tumorigenesis. Using quantitative RT-PCR (qRT-PCR) and Methylation-specific PCR (MSP) assays, we demonstrated that PAX3 was frequently down-regulated by promoter methylation in both primary thyroid cancer tissues and thyroid cancer cell lines. In addition, our data showed that ectopic expression of PAX3 dramatically inhibited thyroid cancer cell proliferation, colony formation, migration and invasion, induced cell cycle arrest and apoptosis and retarded tumorigenic potential in nude mice. Mechanically, PAX3 exerted its tumor suppressor function by inhibiting the activity of major signaling pathways including the phosphatidylinositol-3-kinase (PI3K)/Akt and MAPK/Erk pathways, and enhancing expression and activity of transcription factor FOXO3a. Altogether, our findings provided insight into the role of PAX3 as a novel functional tumor suppressor in thyroid cancer through modulating the activities of PI3K/Akt and MAPK signaling pathways and transcription factor FOXO3a, and demonstrated that epigenetic alterations such as promoter methylation should be a major mechanism of PAX3 inactivation in this cancer.

Site-specific Hypermethylation of RUNX3 Predicts Poor Prognosis in Gastric Cancer

BACKGROUND AND AIMS Methylation status of RUNX3 remains largely unknown in gastric cancer (GC). The aim of this study was to prognostically evaluate the methylation level of CpG sites within RUNX3 promoter region in GC. METHODS Using pyrosequencing, we quantitatively explored the methylation status of 8 CpG sites within RUNX3 promoter region for 76 gastric cancer and 24 normal gastric tissues. We then analyzed the association between methylation level of each CpG site and clinicopathological characteristics and outcomes in the cohort. RESULTS Methylation of RUNX3 promoter was significantly higher in GC than normal subjects. Overall methylation level was closely associated with tumor invasion and TNM stage. Positive associations were found between hypermethylation of the following concerned sites and variables: site -1392, -1397, -1403, -1415 and tumor invasion, as well as TNM stage; site -1392 and lymph node metastasis along with number of lymph node metastases; site -1415 and cancer recurrence; site -1403, -1415 and cancer-related deaths. In multivariate analysis, tumor invasion was correlated with sites -1392 and -1397. Lymph node metastasis was associated with site -1392. Most importantly, methylation of site -1415 was associated with poor survival by using Cox survival regression. CONCLUSION Analysis of RUNX3 gene promoter by quantitative pyrosequencing suggested methylation status of RUNX3 is different in normal and tumor tissues. RUNX3 methylation level is associated with GC, especially the methylation at site -1415 contributes to the poor prognosis in GC. Thus, RUNX3 methylation may serve as a valuable diagnostic and prognostic biomarker in GC.

Proteasome inhibitor MG132 induces thyroid cancer cell apoptosis by modulating the activity of transcription factor FOXO3a

Proteasome inhibitors are promising antitumor drugs with preferable cytotoxicity in malignant cells and have exhibited clinical efficiency in several hematologic malignancies. P53-dependent apoptosis has been reported to be a major mechanism underlying. However, apoptosis can also be found in cancer cells with mutant-type p53, suggesting the involvement of p53-independent mechanism. Tumor suppressor forkhead Box O3 is another substrate of proteasomal degradation, which also functions partially through inducing apoptosis. The aim of this study was to explore the effect of proteasome inhibition on the expression and activity of forkhead Box O3 in thyroid cancer cells. Using flow cytometry, western blot, immunofluorescence staining and quantitative RT-PCR assays, we assessed proteasome inhibitor MG132-induced apoptosis in thyroid cancer cells and its effect on the expression and activity of forkhead Box O3. The resulted showed that MG132 induced significant apoptosis, and caused the accumulation of p53 protein in both p53 wild-type and mutant-type thyroid cancer cell lines, whereas the proapoptotic targets of p53 were transcriptionally upregulated only in the p53 wild-type cells. Strikingly, upon MG132 administration, the accumulation and nuclear translocation of transcription factor forkhead Box O3 as well as transcriptional upregulation of its proapoptotic target genes were found in thyroid cancer cells regardless of p53 status. Cell apoptosis was enhanced by ectopic overexpression while attenuated by silencing of forkhead Box O3. Altogether, we demonstrated that proteasome inhibitor MG132 induces thyroid cancer cell apoptosis at least partially through modulating forkhead Box O3 activity.

Positive Feedback Loops between NrCAM and Major Signaling Pathways Contribute to Thyroid Tumorigenesis

Context: Although neuronal cell adhesion molecule (NrCAM) has been reported to be overexpressed in papillary thyroid cancer (PTCs), its role in this disease remains largely unclear. Objectives: The aim of this study was to explore the biological functions of NrCAM and its potential as a diagnostic marker and therapeutic target in thyroid cancer. Experimental Design: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate messenger RNA expression of investigated genes. The functions of knockdown and ectopic expression of NrCAM in thyroid cancer cells were determine by a series of in vitro and in vivo experiments. Results: We found that NrCAM was highly expressed in PTCs and demonstrated that NrCAM might be a potential marker for preoperative diagnosis of PTC. Moreover, NrCAM depletion dramatically inhibited thyroid cancer cell growth, invasiveness and tumorigenic potential in nude mice. On the other hand, ectopic expression of NrCAM significantly enhanced its tumor-promoting effects. Mechanically, NrCAM exerted its oncogenic function by activating MAPK/Erk and PI3K/Akt pathways via its ectodomain shedding and binding to EGFR and &agr;4&bgr;1 integrins. In turn, these 2 pathways were also responsible for NrCAM overexpression through GSK3&bgr;/&bgr;-catenin signaling axis in thyroid cancer cells. Similar results were also found in transgenic mice with thyroid-specific knock-in of oncogenic BrafV600E (TPO-Cre/LSL-BrafV600E). Conclusions: Our data first reveal positive feedback loops between NrCAM and major signaling pathways contributing to thyroid tumorigenesis by modulating tumor microenvironment, and suggest that NrCAM may represent a potential diagnostic marker and therapeutic target for thyroid cancer.

Increased expression of EHF contributes to thyroid tumorigenesis through transcriptionally regulating HER2 and HER3

E26 transformation-specific (ETS) transcription factor EHF plays a tumor suppressor role in prostate cancer and esophageal squamous cell carcinoma (ESCC), whereas it is overexpressed and may act as an oncogene in ovarian and mammary cancers. However, its biological role in thyroid cancer remains totally unknown. The aim of this study was to explore the biological functions of EHF and its potential as a therapeutic target in thyroid cancer. Using quantitative RT-PCR (qRT-PCR) assay, we evaluated mRNA expression of EHF in a cohort of primary papillary thyroid cancers (PTCs) and matched non-cancerous thyroid tissues. The functions of knockdown and ectopic expression of EHF in thyroid cancer cells were determine by a series of in vitro and in vivo experiments. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to identify its downstream targets. Our data showed that EHF expression was significantly increased in PTCs compared with matched non-cancerous thyroid tissues. EHF knockdown significantly inhibited thyroid cancer cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice and induced cell cycle arrested and apoptosis by modulating the PI3K/Akt and MAPK/Erk signaling pathways. On the other hand, ectopic expression of EHF in thyroid cancer cells notably promoted cell growth and invasiveness. Importantly, EHF was identified as a new transcription factor for HER2 and HER3, contributing to thyroid tumorigenesis. Altogether, our findings suggest that EHF is a novel functional oncogene in thyroid cancer by transcriptionally regulating HER2 and HER3, and may represent a potential therapeutic target for this cancer.

Gender-related differences in the association between concomitant amplification of AIB1 and HER2 and clinical outcomes in glioma patients

BACKGROUND Previous studies demonstrated that AIB1 or HER2 copy number gain (CNG), respectively, were independent predictors for poor prognosis of glioma patients, especially in females. We hypothesize that there are some connections between the two genes and sex-specific characteristics, thus this study aimed to analyze gender-related differences in the prognosis of glioma patients. METHODS Using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) method, we examined AIB1 and HER2 CNG in gliomas samples (n = 114), and inspected the correlation of various genotypes with patients outcomes. RESULTS Concomitant AIB1 and HER2 amplification were closely related to shorter survival time and radiotherapy resistance in female gliomas patients (P < 0.01), which also served as an independent risk factor. No significant prognostic value was found with AIB1 and HER2 CNG in male patients. However, linear regression analysis showed a positive relationship between the copy number of AIB1 and HER2 (P < 0.01) in male patients, rather than female patients. CONCLUSION In this study, we reveal a gender difference in the prognostic value of concomitant AIB1 and HER2 CNG in glioma patients which were barely noticed before. These observations indicated that genetic alterations synergistic with essential respects of sex determination influence glioma biology and patients outcomes.

Turning a Luffa Protein into a Self-Assembled Biodegradable Nanoplatform for Multitargeted Cancer Therapy

The peptide-derived self-assembly platform has attracted increasing attention for its great potential to develop into multitargeting nanomedicines as well as its inherent biocompatibility and biodegradability. However, their clinical application potentials are often compromised by low stability, weak membrane penetrating ability, and limited functions. Herein, inspired by a natural protein from the seeds of Luffa cylindrica, we engineered via epitope grafting and structure design a hybrid peptide-based nanoplatform, termed Lupbin, which is capable of self-assembling into a stable superstructure and concurrently targeting multiple protein-protein interactions (PPIs) located in cytoplasm and nuclei. We showed that Lupbin can efficiently penetrate cell membrane, escape from early endosome-dependent degradation, and subsequently disassemble into free monomers with wide distribution in cytosol and nucleus. Importantly, Lupbin abrogated tumor growth and metastasis through concurrent blockade of the Wnt/β-catenin signaling and reactivation of the p53 signaling, with a highly favorable in vivo biosafety profile. Our strategy expands the application of self-assembled nanomedicines into targeting intercellular PPIs, provides a potential nanoplatform with high stability for multitargeted cancer therapy, and likely reinvigorates the development of peptide-based therapeutics for the treatment of different human diseases including cancer.

Identification of MAP kinase pathways as therapeutic targets in gallbladder carcinoma using targeted parallel sequencing

The aim of this study was to profile somatic mutation spectrum in gallbladder cancers (GBCs), and determine the role of MAP kinase pathway in GBC by a series of in vitro and in vivo studies. We performed targeted massively parallel sequencing of DNA isolated from GBCs and matched blood from 14 GBC patients to search for mutations in 504 genes commonly altered in human cancers. We identified recurrent mutations enriched in several major signaling pathways including MAP kinase, Wnt/β-catenin and NF-κB pathways. Immunohistochemistry analysis further validated overactivation of MAP kinase and Wnt pathways in a panel of GBC samples. By treating GBC cells with MEK inhibitor trametinib, we found that trametinib not only dramatically inhibited the activity of MAPK/ERK pathway, but also blocked the Wnt/β-catenin signaling through decreasing β-catenin expression or suppressing nucleus translocation of β-catenin. Moreover, trametinib inhibited the proliferation of GBC cell in a dose- and time-dependent manner, induced GBC cell apoptosis, and inhibited GBC cell migration and invasion. Growth of xenograft tumors derived from GBC cell line NOZ in nude mice was also significantly inhibited by trametinib. Our data highlight the critical role of MAP kinase pathways in GBC pathogenesis, and may represent therapeutic targets for this cancer.