Lijia Dong

Identification of genomic alterations in oesophageal squamous cell cancer

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

Overexpression of Aurora-A Contributes to Malignant Development of Human Esophageal Squamous Cell Carcinoma

Purpose: Aurora-A/STK15/BTAK, a centrosome-associated oncogenic protein, is implicated in the control of mitosis. Overexpression of Aurora-A has been shown to result in chromosomal aberration and genomic instability. Multiple lines of evidence indicate that Aurora-A induces cell malignant transformation. In the current study, we are interested in investigating the expression of Aurora-A in human esophageal squamous cell carcinoma (ESCC) and characterizing the association of Aurora-A with ESCCmalignant progression. Experimental Design: Aurora-A protein expression was examined in 84 ESCC tissues and 81 paired normal adjacent tissues by either immunohistochemistry or Western blot analysis. In addition, a gene-knockdown small interfering RNA technique was used in ESCC cells to investigate whether Aurora-A contributes to the ability of a tumor to grow invasively. Results: The amount of Aurora-A protein in ESCC was considerably higher than that in normal adjacent tissues. Overexpression of Aurora-A was observed in 57 of 84 (67.5%) ESCC samples. In contrast, <2% of normal adjacent tissue displayed high expression of Aurora-A. Interestingly, overexpression of Aurora-A seemed to correlate with the invasive malignancy of ESCC. Disruption of endogenous Aurora-A using small interfering RNA technique substantially suppressed cell migrating ability. Conclusion: The findings presented in this report show that Aurora-A expression is elevated in human esophageal squamous cell carcinoma and is possibly associated with tumor invasion, indicating that overexpression of Aurora-A may contribute to ESCC occurrence and progression.

Integrated ionization approach for RRLC-MS/MS-based metabonomics: finding potential biomarkers for lung cancer.

An integrated ionization approach of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) combining with rapid resolution liquid chromatography mass spectrometry (RRLC-MS) has been developed for performing global metabonomic analysis on complex biological samples. This approach was designed to overcome the low ionization efficiencies of endogenous metabolites due to diverse physicochemical properties as well as ion suppression, and obtain comprehensive metabolite profiles in LC-MS analysis. Ionization capability and applicability were manifested by improved ionization efficiency and enlarged metabolite coverage in analysis on typical urinary metabolite standards and urine samples from healthy volunteers. The method was validated by the limit of detection and precision. When applied to the global metabonomic studies of lung cancer, more comprehensive biomarker candidates were obtained to reflect metabolic traits between healthy volunteers and lung cancer patients, including 74 potential biomarkers in positive ion mode and 59 in negative ion mode. Taking identical potential biomarkers of any two or three ionization methods into account, analysis using ESI-MS in positive (+) and negative (-) ion mode contributed to 70 and 64% of the total potential biomarkers, respectively. The biomarker discovery capability of (+/-) APCI-MS accounted for 45 and 42% of the overall; meanwhile (+/-) APPI-MS amounted for 39 and 54%. These results indicated that potential biomarkers with vital biological information could be missed if only a single ionization method was used. Furthermore, 11 potential biomarkers were identified including amino acids, nucleosides, and a metabolite of indole. They revealed elevated amino acid and nucleoside metabolism as well as protein degradation in lung cancer patients. This proposed approach provided a more comprehensive picture of the metabolic changes and further verified identical biomarkers that were obtained simultaneously using different ionization methods.

Global and Targeted Metabolomics of Esophageal Squamous Cell Carcinoma Discovers Potential Diagnostic and Therapeutic Biomarkers

Diagnostic and therapeutic biomarkers useful for esophageal squamous cell carcinoma (ESCC) have the ability to increase the long term survival of cancer patients. A metabolomics study, using plasma from four groups including ESCC patients before, during, and after chemoradiotherapy (CRT) and healthy controls, was originally carried out by LC-MS to determine global alterations in the metabolic profiles and find biomarkers potentially applicable to diagnosis and monitoring treatment effects. It is worth pointing out that a clear clustering and separation of metabolic data from the four groups was observed, which indicated that disease status and treatment intervention resulted in specific metabolic perturbations in the patients. A series of metabolites were found to be significantly altered in ESCC patients versus healthy controls and in pre- versus post-treatment patients based on multivariate statistical data analysis (MVDA). To further validate the reliability of these potential biomarkers, an independent validation was performed by using the selected reaction monitoring (SRM) based targeted approach. Finally, 18 most significantly altered plasma metabolites in ESCC patients, relative to healthy controls, were tentatively identified as lysophosphatidylcholines (lysoPCs), fatty acids, l-carnitine, acylcarnitines, organic acids, and a sterol metabolite. The classification performance of these metabolites were analyzed by receiver operating characteristic (ROC)1 analysis and a biomarker panel was generated. Together, biological significance of these metabolites was discussed. Comparison between pre- and post-treatment patients generated 11 metabolites as potential therapeutic biomarkers that were tentatively identified as amino acids, acylcarnitines, and lysoPCs. Levels of three of these (octanoylcarnitine, lysoPC(16:1), and decanoylcarnitine) were closely correlated with treatment effect. Moreover, variation of these three potential biomarkers was investigated over the treatment course. The results suggest that these biomarkers may be useful in diagnosis, as well as in monitoring therapeutic responses and predicting outcomes of the ESCC.

RRLC-MS/MS-based metabonomics combined with in-depth analysis of metabolic correlation network: finding potential biomarkers for breast cancer

A metabonomics strategy based on rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS), multivariate statistics and metabolic correlation networks has been implemented to find biologically significant metabolite biomarkers in breast cancer. RRLC-MS/MS analysis by electrospray ionization (ESI) in both positive and negative ion modes was employed to investigate human urine samples. The resulting data matrices were analyzed using multivariate analysis. Application of orthogonal projections to latent structures discriminate analysis (OPLS-DA) allowed us to extract several discriminated metabolites reflecting metabolic characteristics between healthy volunteers and breast cancer patients. Correlation network analysis between these metabolites has been further applied to select more reliable biomarkers. Finally, high resolution MS and MS/MS analyses were performed for the identification of the metabolites of interest. We identified 12 metabolites as potential biomarkers including amino acids, organic acids, and nucleosides. They revealed elevated tryptophan and nucleoside metabolism as well as protein degradation in breast cancer patients. These studies demonstrate the advantages of integrating metabolic correlation networks with metabonomics for finding significant potential biomarkers: this strategy not only helps identify potential biomarkers, it also further confirms these biomarkers and can even provide biochemical insights into changes in breast cancer.

Gadd45a Interacts with Aurora-A and Inhibits Its Kinase Activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21waf1/cip1, which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.

Migfilin Protein Promotes Migration and Invasion in Human Glioma through Epidermal Growth Factor Receptor-mediated Phospholipase C-γ and STAT3 Protein Signaling Pathways

Background: The oncogenesis and developmental mechanisms of glioma must be clarified to control the disease. Results: Migfilin relates to pathological grades, prognosis of glioma, and regulates motility of glioma cells. Conclusion: Migfilin mediates migration and invasion through EGFR-induced PLC-γ and STAT3 pathways. Significance: Migfilin helps us better understand the pathogenesis of glioma, and Migfilin may be a molecular marker in diagnosis and an indicator in prognosis. Migfilin is critical for cell shape and motile regulation. However, its pathological role in glioma is unknown. Using an immunohistochemical staining assay, we demonstrate that there is a significant correlation between expression of Migfilin and pathological tumor grade in 217 clinical glioma samples. High Migfilin expression is associated with poor prognosis for patients with glioma. Investigation of the molecular mechanism shows that Migfilin promotes migration and invasion in glioma cells. Moreover, Migfilin positively modulates the expression and activity of epidermal growth factor receptor, and Migfilin-mediated migration and invasion depend on epidermal growth factor receptor-induced PLC-γ and STAT3-signaling pathways. Our results may provide significant clinical application, including use of Migfilin as a molecular marker in glioma for early diagnosis and as an indicator of prognosis.

Stable knockdown of Aurora-A by vector-based RNA interference in human esophageal squamous cell carcinoma cell line inhibits tumor cell proliferation, invasion and enhances apoptosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers, and the existing treatment approaches have not been able to effectively manage this dreaded cancer. Therefore, continuing efforts are ongoing to explore novel targets and strategies for the management of ESCC. It has been shown that amplification and overexpression of the Aurora-A occur in several types of human tumors, including ESCC. Moreover, overexpression of Aurora-A is shown to associate with the grades of tumor differentiation and metastasis of ESCC. These render Aurora-A an interesting target for antitumor therapy. Recently, vector-based RNA interference (RNAi) expression systems have been successfully used to silence gene expression, but knockdown of Aurora-A by vector-based RNAi as a therapeutic model for ESCC treatment is not fully established. In this study, we used a DNA vector-based RNAi approach by expressing short hairpin RNA (shRNA) to knock down Aurora-A. Western blotting analysis and reverse transcription PCR (RT-PCR) showed that expressions of Aurora-A were efficiently downregulated at both the protein and mRNA levels by stable transfection with Aurora-A siRNA expression vector. The stable suppression of Aurora-A expression inhibited the proliferation and invasion of EC9706 cells. Furthermore, when Aurora-A was stably downregulated, cisplatin-induced cytotoxic effects and apoptosis were increased dramatically. These data indicate that vector-mediated silencing of the Aurora-A gene may provide new avenues toward the study of the role of Aurora-A overexpression in tumor cells. Make a novel therapeutic approach to treatment of ESCC and other malignant tumors overexpressing Aurora-A.

Analysis of multiplex endogenous estrogen metabolites in human urine using ultra-fast liquid chromatography-tandem mass spectrometry: a case study for breast cancer.

A rapid, sensitive, specific and accurate analytical method of ultra-fast liquid chromatography combined with tandem mass spectrometry (UFLC-MS/MS) was established for simultaneous quantitative analysis of 16 distinct endogenous estrogens and their metabolites (EMs) in postmenopausal female urine. The quantitative method utilized a hydrolysis/extraction/derivatization step and a UFLC system to achieve separation in 16 min. The lower limit of quantitation for each estrogen metabolite was 2 pg mL(-1) with the percent recovery of a known added amount of estrogen at 93.2-109.3%. The intra-batch accuracy and precision for all analytes were 87.5-107.7% and 0.6-11.7%, respectively, while inter-batch accuracy and precision were 87.0-105.8% and 1.2-10.2%, respectively. Using this developed and validated method, the comprehensive metabolic profiling of 16 EMs in urine samples of 86 postmenopausal female breast cancer patients and 36 healthy controls was investigated by systematic statistical analysis. As a result, the circulating levels of 6 EMs were found to be different by a comparison of patients and healthy controls. The parent estrogens, estrone (E1) and 17β-estradiol (E2), as well as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) were produced in higher abundance, whereas 16α-hydroxyestrone (16α-OHE1) and 2-methoxyestradiol (2-MeOE2) were decreased in the breast cancer group. 2-OHE2 and 4-OHE2 in particular showed significant elevation in patients, which are consistent with the carcinogenic mechanism hypothesis that catechol estrogens can react with DNA via quinones, resulting in mutations to induce breast cancer. Thus, 2,4-hydroxylation may be the dominant metabolic pathway for parent estrogens rather than 16α-hydroxylation. The lower level of 2-MeOE2 in the breast cancer group was believed to correlate with its protective effect against tumor formation. This study could provide valuable information on the association of the EM metabolic pathway with carcinogenesis as well as identify potential biomarkers for estrogen-induced breast cancer risk.

Dasatinib enhances cisplatin sensitivity in human esophageal squamous cell carcinoma (ESCC) cells via suppression of PI3K/AKT and Stat3 pathways

The clinical efficacy of cisplatin in esophageal squamous cell carcinoma (ESCC) treatment remains undesirable. Src, a non-receptor tyrosine kinase involved in multiple fields of tumorigenesis, recently has been indicated as a promising therapeutic target in the treatment of solid tumors including ESCC. However, whether inhibition of Src activity can increase cisplatin efficacy in ESCC cells remains unknown. The present study found that inhibition of Src by its inhibitor-dasatinib sensitized ESCC cells to cisplatin in vitro. Our data also suggest a likely mechanism for this synergy that dasatinib reduces expression of critical oncogenic members of the signaling pathways, such as AKT or Stat3, and cisplatin-resistant molecules, such as ERCC1 and BRCA1, under the control of Src. Furthermore, dasatinib could sensitize ESCC cells to another platin-based agent, carboplatin. Therefore, this study provides a potential target for improving cisplatin efficacy in ESCC therapy.