Jiangxue Wu

Genetic and Epigenetic Down-regulation of MicroRNA-212 Promotes Colorectal Tumor Metastasis via Dysregulation of MnSOD

BACKGROUND & AIMS Altered functions of microRNAs (miRNAs) have been associated with colorectal cancer (CRC). miR-212 is transcribed from a stable intron of a non-protein coding gene, and is reportedly down-regulated in different tumor types. We investigated the role of miR-212 in colorectal carcinogenesis and progression. METHODS We analyzed the expression of miR-212 by real-time polymerase chain reaction (PCR) analysis of colorectal cell lines and 180 paired tumor samples and surrounding healthy tissue. We overexpressed and knocked down miR-212 in CRC cell lines and assessed the in vitro effects. We also studied the effects of miR-212 overexpression on metastasis of tumors grown from HCT116 cells in nude mice. RESULTS Overexpression of miR-212 inhibited CRC cell migration and invasion in vitro and formation of intrahepatic and pulmonary metastasis in vivo. We identified manganese superoxide dismutase (MnSOD) messenger RNA as a direct target of miR-212, and observed an inverse correlation between the level of miR-212 and MnSOD protein in colorectal tumor samples. MnSOD was required for down-regulation of epithelial markers and up-regulation of mesenchymal markers in CRC cells, indicating that it promoted the epithelial-mesenchymal transition. Overexpression of miR-212 reduced the levels of MnSOD to block the epithelial-mesenchymal transition process. Loss of heterozygosity and promoter hypermethylation each contributed to the down-regulation of miR-212. Reduced levels of miR-212 were associated with a more aggressive tumor phenotype and short disease-free survival times of patients (P = .0045; overall survival, P = .0015). CONCLUSIONS miR-212 is down-regulated in human CRC tissues via genetic and epigenetic mechanisms. miR-212 might prevent tumor progression by targeting MnSOD messenger RNA; reduction of miR-212 could be a prognostic marker for patients with CRC. miR-212 and MnSOD might also be therapeutic targets for cancer.

Minicircle-IFNγ induces antiproliferative and antitumoral effects in human nasopharyngeal carcinoma

Purpose: The aims of this work were to investigate the antitumor effect of IFNγ gene transfer on human nasopharyngeal carcinoma (NPC) and to assess the potential of minicircle vector for antitumor gene therapy. Experimental Design: We developed a recombinant minicircle vector carrying the human IFNγ gene and evaluated the effects of minicircle-mediated IFNγ gene transfer on NPC cell lines in vitro and on xenografts in vivo. Results: Relative to p2ΦC31-IFNγ, minicircle-mediated IFNγ gene transfer in vitro resulted in 19- to 102-fold greater IFNγ expression levels in transfected cells (293, NIH 3T3, CNE-1, CNE-2, and C666-1) and inhibited the growth of CNE-1, CNE-2, and C666-1 cells more efficiently, reducing relative growth rates to 7.1 ± 1.6%, 2.7 ± 1.0%, and 6.1 ± 1.6%, respectively. Flow cytometry and caspase-3 activity assays suggested that the antiproliferative effects of IFNγ gene transfer on NPC cell lines could be attributed to G0-G1 arrest and apoptosis. Minicircle-mediated intratumoral IFNγ expression in vivo was 11 to 14 times higher than p2ΦC31-IFNγ in CNE-2- and C666-1-xenografted mice and lasted for 21 days. Compared with p2ΦC31-IFNγ treatment, minicircle-IFNγ treatment significantly increased survival and achieved inhibition rates of 77.5% and 83%, respectively. Conclusions: Our data indicate that IFNγ gene transfer exerts antiproliferative effects on NPC cells in vitro and leads to a profound antitumor effect in vivo. Minicircle-IFNγ is more efficient than corresponding conventional plasmids due to its capability of mediating long-lasting high levels of IFNγ gene expression. Therefore, minicircle-mediated IFNγ gene transfer is a promising novel approach in the treatment of NPC.

Overexpression of Sirt7 Exhibits Oncogenic Property and Serves as a Prognostic Factor in Colorectal Cancer

Purpose: Sirtuins play an important role in cancer development. Sirt7, as a member of this family, is frequently overexpressed in certain carcinomas, but the oncogenic mechanism is seldom reported. In this study, Sirt7 was characterized for its role in colorectal cancer aggressiveness and underlying molecular mechanisms. Experimental Design: Quantitative PCR, Western blotting, and immunohistochemistry were performed to study Sirt7 expression in a cohort of colorectal cancer tissues and non-tumor tissues and cells. A series of in vitro and in vivo assays was performed to elucidate the function of Sirt7 in colorectal cancer and its underlying mechanisms. Association between the Sirt7 signature and survival was examined using Kaplan–Meier analysis and log-rank tests. Results: The Sirt7 protein level significantly correlated with tumor stage (P = 0.029), lymph node metastasis (P = 0.046), and poor patient survival (P < 0.05). Sirt7 knockdown significantly inhibited colorectal cancer cell proliferation, colony formation, and motility. Ectopic Sirt7 expression promoted colony formation, induced a more invasive phenotype, and accelerated cell growth both in vitro and in vivo. Moreover, Sirt7 enhanced MAPK pathway activity concomitantly with p-ERK and p-MEK upregulation. In Sirt7-overexpressing cells, the mesenchymal markers vimentin and fibronectin were upregulated, and the epithelial markers E-cadherin and β-catenin were downregulated, which was linked to enhanced invasion by colorectal cancer cells. Conclusion: Our findings suggest that Sirt7 plays an important role in the development and progression of human colorectal cancer and functions as a valuable marker of colorectal cancer prognosis. Clin Cancer Res; 20(13); 3434–45. ©2014 AACR.

OTUB1 promotes metastasis and serves as a marker of poor prognosis in colorectal cancer

BackgroundOTUB1 (OTU deubiquitinase, ubiquitin aldehyde binding 1) is a deubiquitinating enzyme (DUB) that belongs to the OTU (ovarian tumor) superfamily. The aim of this study was to clarify the role of OTUB1 in colorectal cancer (CRC) and to identify the mechanism underlying its function.MethodsTwo hundred and sixty CRC samples were subjected to association analysis of OTUB1 expression and clinicopathological variables using immunohistochemical (IHC) staining. Overexpression of OTUB1 was achieved in SW480 and DLD-1 cells, and downregulation of OTUB1 was employed in SW620 cells. Then, migration and invasion assays were performed, and markers of the epithelial-mesenchymal transition (EMT) were analyzed. In addition, hepatic metastasis models in mice were used to validate the function of OTUB1 in vivo.ResultsOTUB1 was overexpressed in CRC tissues, and the expression level of OTUB1 was associated with metastasis. A high expression level of OTUB1 was also associated with poor survival, and OTUB1 served as an independent prognostic factor in multivariate analysis. OTUB1 also promoted the metastasis of CRC cell lines in vitro and in vivo by regulating EMT.ConclusionsOTUB1 promotes CRC metastasis by facilitating EMT and acts as a potential distant metastasis marker and prognostic factor in CRC. Targeting OTUB1 may be helpful for the treatment of CRC.

Induction of cell cycle arrest and apoptosis in human nasopharyngeal carcinoma cells by ZD6474, an inhibitor of VEGFR tyrosine kinase with additional activity against EGFR tyrosine kinase

ZD6474 is a vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. The present study was undertaken to investigate the direct antiproliferative effect of ZD6474 on human nasopharyngeal carcinoma (NPC) in vitro and the antitumor activity on NPC xenografts in vivo. Results indicated that ZD6474 treatment inhibited EGFR phosphorylation and led to a dose‐ and time‐dependent decrease in NPC cell (CNE‐1, CNE‐2 and C666‐1) proliferation. Further investigation demonstrated G0/G1 cell cycle arrest in all 3 cell lines, which was associated with an upregulation of p21 and/or p27, and downregulation of CDK4, CDK6 and CDK2. ZD6474 treatment also induced apoptosis in CNE‐1 and CNE‐2 cells. The apoptosis mechanisms involved reduction of Bcl‐2 and/or Bcl‐XL, induction of Bak and/or Bax, and activation of caspases‐3, ‐9 and/or ‐8. The in vivo antitumor activity was evaluated in CNE‐2 and C666‐1 xenografted nude mice. Administration of ZD6474 (25–100 mg/kg/day, once‐daily, p.o.) produced a dose‐dependent inhibition of tumor growth and prolonged survival in both models. This study suggests that ZD6474 exerts direct antiproliferative effects on NPC cell lines in vitro by inducing G0/G1 arrest and apoptosis, and potent antitumor effects on NPC xenografts in vivo. It indicates that ZD6474 may offer a new and effective treatment for human NPC.

Multicenter Randomized Phase 2 Clinical Trial of a Recombinant Human Endostatin Adenovirus in Patients with Advanced Head and Neck Carcinoma

A randomized, open-label, phase 2, multicenter clinical trial was conducted to evaluate the efficacy and safety of the addition of a recombinant human endostatin adenovirus (E10A) to cisplatin and paclitaxel in patients with advanced head and neck squamous cell carcinoma or nasopharyngeal carcinoma. Patients with locally advanced or metastatic head and neck squamous cell carcinoma or nasopharyngeal carcinoma not suitable for operation or radiotherapy were randomly assigned to receive E10A plus chemotherapy every 3 weeks for a maximum of six cycles or to receive chemotherapy only. One hundred and thirty-six eligible patients were randomly assigned. The addition of E10A did not significantly improve the objective response rate (29.9 versus 39.7%, P = 0.154). However, patients who received endostatin had longer progression-free survival (7.03 versus 3.60 months, P = 0.006; hazard ratio: 0.55). The combination of E10A with chemotherapy benefited prior chemotherapy-treated patients and those who received three to four treatment cycles (6.50 versus 3.43 months, P = 0.003; 8.27 versus 4.27 months, P = 0.018; respectively). The overall disease control rate significantly increased from 80.6% in the control group to 92.6% in the test group (P = 0.034). Except for fever, no adverse events were associated with the E10A treatment. In summary, E10A plus chemotherapy is a safe and effective therapeutic approach in patients with advanced head and neck squamous cell carcinoma or nasopharyngeal carcinoma.

Cryptochrome 1 Overexpression Correlates with Tumor Progression and Poor Prognosis in Patients with Colorectal Cancer

Background Clock genes drive about 5–15% of genome-wide mRNA expression, and disruption of the circadian clock may deregulate the cells normal biological functions. Cryptochrome 1 is a key regulator of the circadian feedback loop and plays an important role in organisms. The present study was conducted to investigate the expression of Cry1 and its prognostic significance in colorectal cancer (CRC). In addition, the function of Cry1 in human CRC was investigated in cell culture models. Methods Real-time quantitative PCR, Western blot analysis and immunohistochemistry were used to explore Cry1 expression in CRC cell lines and primary CRC clinical specimens. MTT and colony formation assays were used to determine effects on cellular proliferation ability. The animal model was used to explore the Cry1 impact on the tumor cellular proliferation ability in vivo. Transwell assays were performed to detect the migration ability of the cell lines. Statistical analyzes were applied to evaluate the diagnostic value and the associations of Cry1 expression with clinical parameters. Results Cry1 expression was up regulated in the majority of the CRC cell lines and 168 primary CRC clinical specimens at the protein level. Clinical pathological analysis showed that Cry1 expression was significantly correlated with lymph node metastasis (p = 0.004) and the TNM stage (p = 0.003). High Cry1 expression was associated with poor overall survival in CRC patients (p = 0.010). Experimentally, we found that up-regulation of Cry1 promoted the proliferation and migration of HCT116 cells, while down-regulation of Cry1 inhibited the colony formation and migration of SW480 cells. Conclusions These results suggest that Cry1 likely plays important roles in CRC development and progression andCry1 may be a prognostic biomarker and a promising therapeutic target for CRC.

Minicircle-oriP-IFNγ: A Novel Targeted Gene Therapeutic System for EBV Positive Human Nasopharyngeal Carcinoma

Background Nonviral vectors are attractively used for gene therapy owing to their distinctive advantages. Our previous study has demonstrated that transfer of human IFNγ gene into nasopharyngeal carcinoma (NPC) by using a novel nonviral vector, minicircle (mc), under the control of cytomegalovirus (CMV) promoter was effective to inhibit tumor growth. However, therapies based on CMV promoter cannot express the targeted genes in cancer tissues. Previous studies indicated that the development of human NPC was closely associated with Epstein-Barr virus (EBV) and demonstrated the transcriptional enhancer function of oriP when bound by EBV protein. Therefore, the present study is to explore the targeted gene expression and the anti-tumor effect of a novel tumor-specific gene therapeutic system (mc-oriP-IFNγ) in which the transgene expression was under the transcriptional regulation of oriP promoter. Methodology/Principal Findings Dual-luciferase reporter assay and ELISA were used to assess the expression of luciferase and IFNγ. WST assay was used to assess the cell proliferation. RT-PCR was used to detect the mRNA level of EBNA1. RNAi was used to knockdown the expression of EBNA1. NPC xenograft models in nude mice were used to investigate the targeted antitumor efficacy of mc-oriP-IFNγ. Immunohistochemistry was used to detect the expression and the activity of the IFNγ in tumor sections. Our results demonstrated that mc-oriP vectors mediated comparable gene expression and anti-proliferative effect in the EBV-positive NPC cell line C666-1 compared to mc-CMV vectors. Furthermore, mc-oriP vectors exhibited much lower killing effects on EBV-negative cell lines compared to mc-CMV vectors. The targeted expression of mc-oriP vectors was inhibited by EBNA1-siRNA in C666-1. This selective expression was corroborated in EBV-positive and -negative tumor models. Conclusions/Significance This study demonstrates the feasibility of mc-oriP-IFNγ as a safe and highly effective targeted gene therapeutic system for the treatment of EBV positive NPC.

Genistein Induces Growth Inhibition and G2/M Arrest in Nasopharyngeal Carcinoma Cells

Nasopharyngeal carcinoma (NPC) is an endemic malignant disease of the head and neck region with unique features including striking ethnic and geographic variations as well as multifactorial etiology. Previous studies have demonstrated the anticancer properties of genistein, the major soy isoflavonoid, in several human cancer cells such as breast, prostate, colon, gastric, lung, and hepatoma. However, the action of genistein in NPC cells has not been determined. In this study, we investigated the inhibitory effects of genistein on NPC cells and its possible underlying mechanisms. We found that genistein dose-dependently inhibited the proliferation of human NPC cell line CNE2 cells. DNA flow cytometric analysis revealed that 30 to 120 μM genistein induced dramatic G2/M phase arrest in NPC cells. The mRNA expression levels, as shown by gene expression array and quantitative real-time polymerase chain reaction, and the protein expression levels of the cell cycle regulators p21Cip1 and ATR (Ataxia telangiectasia and Rad3 related) were elevated following genistein treatment. Interestingly, we also observed concomitant induction of p15Ink4b in genistein induced inhibitory effects in NPC cells. Moreover, selective estrogen receptor modulators did not affect genistein induced growth inhibition. These findings provide new insights into the potential intervention of NPC with genistein.

Downregulation of CDC27 inhibits the proliferation of colorectal cancer cells via the accumulation of p21Cip1/Waf1.

Dysregulated cell cycle progression has a critical role in tumorigenesis. Cell division cycle 27 (CDC27) is a core subunit of the anaphase-promoting complex/cyclosome, although the specific role of CDC27 in cancer remains unknown. In our study, we explored the biological and clinical significance of CDC27 in colorectal cancer (CRC) growth and progression and investigated the underlying molecular mechanisms. Results showed that CDC27 expression is significantly correlated with tumor progression and poor patient survival. Functional assays demonstrated that overexpression of CDC27 promoted proliferation in DLD1 cells, whereas knockdown of CDC27 in HCT116 cells inhibited proliferation both in vitro and in vivo. Further mechanistic investigation showed that CDC27 downregulation resulted in G1/S phase transition arrest via the significant accumulation of p21 in HCT116 cells, and the upregulation of CDC27 promoted G1/S phase transition via the attenuation of p21 in DLD1 cells. Furthermore, we also demonstrated that CDC27 regulated inhibitor of DNA binding 1 (ID1) protein expression in DLD1 and HCT116 cells, and rescue assays revealed that CDC27 regulated p21 expression through modulating ID1 expression. Taken together, our results indicate that CDC27 contributes to CRC cell proliferation via the modulation of ID1-mediated p21 regulation, which offers a novel approach to the inhibition of tumor growth. Indeed, these findings provide new perspectives for the future study of CDC27 as a target for CRC treatment.