Man Zhi Li

Bmi-1 Is a Novel Molecular Marker of Nasopharyngeal Carcinoma Progression and Immortalizes Primary Human Nasopharyngeal Epithelial Cells

The Bmi-1 oncoprotein regulates proliferation and oncogenesis in human cells. Its overexpression leads to senescence bypass in human fibroblasts and immortalization of human mammary epithelial cells. In this study, we report that compared with normal nasopharyngeal epithelial cells (NPEC), Bmi-1 is overexpressed in nasopharyngeal carcinoma cell lines. Importantly, Bmi-1 was also found to be overexpressed in 29 of 75 nasopharyngeal carcinoma tumors (38.7%) by immunohistochemical analysis. In contrast to nasopharyngeal carcinoma, there was no detectable expression of Bmi-1 in noncancerous nasopharyngeal epithelium. Moreover, high Bmi-1 expression positively correlated with poor prognosis of nasopharyngeal carcinoma patients. We also report that the overexpression of Bmi-1 leads to bypass of senescence and immortalization of NPECs, which normally express p16(INK4a) and exhibit finite replicative life span. Overexpression of Bmi-1 in NPECs led to the induction of human telomerase reverse transcriptase activity and reduction of p16(INK4a) expression. Mutational analysis of Bmi-1 showed that both RING finger and helix-turn-helix domains of it are required for immortalization of NPECs. Our findings suggest that Bmi-1 plays an important role in the development and progression of nasopharyngeal carcinoma, and that Bmi-1 is a valuable marker for assessing the prognosis of nasopharyngeal carcinoma patients. Furthermore, this study provides the first cellular proto-oncogene immortalized nasopharyngeal epithelial cell line, which may serve as a cell model system for studying the mechanisms involved in the tumorigenesis of nasopharyngeal carcinoma.

The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells

The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1-mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelial-mesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility. Furthermore, upregulation of Bmi-1 led to the stabilization of Snail, a transcriptional repressor associated with EMT, via modulation of PI3K/Akt/GSK-3beta signaling. Chromatin immunoprecipitation assays revealed that Bmi-1 transcriptionally downregulated expression of the tumor suppressor PTEN in tumor cells through direct association with the PTEN locus. This in vitro analysis was consistent with the statistical inverse correlation detected between Bmi-1 and PTEN expression in a cohort of human nasopharyngeal carcinoma biopsies. Moreover, ablation of PTEN expression partially rescued the migratory/invasive phenotype of Bmi-1-silenced cells, indicating that PTEN might be a major mediator of Bmi-1-induced EMT. Our results provide functional and mechanistic links between the oncoprotein Bmi-1 and the tumor suppressor PTEN in the development and progression of cancer.

Bmi-1 promotes invasion and metastasis, and its elevated expression is correlated with an advanced stage of breast cancer

BackgroundB-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) acts as an oncogene in various tumors, and its overexpression correlates with a poor outcome in several human cancers. Ectopic expression of Bmi-1 can induce epithelial-mesenchymal transition (EMT) and enhance the motility and invasiveness of human nasopharyngeal epithelial cells (NPECs), whereas silencing endogenous Bmi-1 expression can reverse EMT and reduce the metastatic potential of nasopharyngeal cancer cells (NPCs). Mouse xenograft studies indicate that coexpression of Bmi-1 and H-Ras in breast cancer cells can induce an aggressive and metastatic phenotype with an unusual occurrence of brain metastasis; although, Bmi-1 overexpression did not result in oncogenic transformation of MCF-10A cells. However, the underlying molecular mechanism of Bmi-1-mediated progression and the metastasis of breast cancer are not fully elucidated at this time.ResultsBmi-1 expression is more pronouncedly increased in primary cancer tissues compared to matched adjacent non-cancerous tissues. High Bmi-1 expression is correlated with advanced clinicopathologic classifications (T, N, and M) and clinical stages. Furthermore, a high level of Bmi-1 indicates an unfavorable overall survival and serves as a high risk marker for breast cancer. In addition, inverse transcriptional expression levels of Bmi-1 and E-cadherin are detected between the primary cancer tissues and the matched adjacent non-cancerous tissues. Higher Bmi-1 levels are found in the cancer tissue, whereas the paired adjacent non-cancer tissue shows higher E-cadherin levels. Overexpression of Bmi-1 increases the motility and invasive properties of immortalized human mammary epithelial cells, which is concurrent with the increased expression of mesenchymal markers, the decreased expression of epithelial markers, the stabilization of Snail and the dysregulation of the Akt/GSK3β pathway. Consistent with these observations, the repression of Bmi-1 in highly metastatic breast cancer cells remarkably reduces cellular motility, invasion and transformation, as well as tumorigenesis and lung metastases in nude mice. In addition, the repression of Bmi-1 reverses the expression of EMT markers and inhibits the Akt/GSK3β/Snail pathway.ConclusionsThis study demonstrates that Bmi-1 promotes the invasion and metastasis of human breast cancer and predicts poor survival.

Epstein-barr virus-encoded LMP2A induces an epithelial- mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.

Genomic sequence analysis of Epstein-Barr virus strain GD1 from a nasopharyngeal carcinoma patient

ABSTRACT To date, the only entire Epstein-Barr virus (EBV) genomic sequence available in the database is the prototype B95.8, which was derived from an individual with infectious mononucleosis. A causative link between EBV and nasopharyngeal carcinoma (NPC), a disease with a distinctly high incidence in southern China, has been widely investigated. However, no full-length analysis of any substrain of EBV from this area has been reported. In this study, we analyzed the entire genomic sequence of an EBV strain from a patient with NPC in Guangdong, China. This EBV strain was termed GD1 (Guangdong strain 1), and the full-length sequence of GD1 was submitted to the GenBank database. The assigned accession number is AY961628 . The entire GD1 sequence is 171,656 bp in length, with 59.5% G+C content and 40.5% A+T content. We detected many sequence variations in GD1 compared to prototypical strain B95.8, including 43 deletion sites, 44 insertion sites, and 1,413 point mutations. Furthermore, we evaluated the frequency of some of these GD1 mutations in Cantonese NPC patients and found them to be highly prevalent. These findings suggest that GD1 is highly representative of the EBV strains isolated from NPC patients in Guangdong, China, an area with the highest incidence of NPC in the world. Furthermore, these findings provide the second full-length sequence analysis of any EBV strain as well as the first full-length sequence analysis of an NPC-derived EBV strain.

Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells.

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for nasopharyngeal carcinoma (NPC). However, drug resistance may occur after several cycles of 5-FU-based chemotherapy. The oncogene B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI-1) has been shown to be involved in the protection of cancer cells from apoptosis. In this study, 5-FU treatment could increase the percentage of apoptotic NPC cells among BMI-1/RNAi-transfected cells than that among cells transfected with the empty vector. The 50% inhibitory concentration (IC(50)) values of 5-FU were significantly decreased to a greater extent in the cells transfected with BMI-1/RNAi. Most importantly, the expression of phospho-AKT and the anti-apoptotic protein BCL-2 were downregulated in the cells in which BMI-1 expression was inhibited, whereas the apoptosis-inducer BAX was observed to be upregulated. Abrogation of AKT pathway by a PI3K inhibitor could not further increase the sensitivity to 5-FU in the cells with reduced BMI-1 expression. Taken together, BMI-1 depletion enhanced the chemosensitivity of NPC cells by inducing apoptosis; which is associated with inhibition of the PI3K/AKT pathway.

Centromere Protein H Is a Novel Prognostic Marker for Nasopharyngeal Carcinoma Progression and Overall Patient Survival

Purpose: The aim of the present study was to analyze the expression of Centromere protein H (CENP-H), one of the fundamental components of the human active kinetochore, in nasopharyngeal carcinoma (NPC) and to correlate it with clinicopathologic data, including patient survival. Experimental Design: Using reverse transcription-PCR and Western blot, we detected the expression of CENP-H in normal nasopharyngeal epithelial cells, immortalized nasopharyngeal epithelial cell lines, and NPC cell lines. Using immunohistochemistry, we analyzed CENP-H protein expression in 160 clinicopathologically characterized NPC cases. Statistical analyses were applied to test for prognostic and diagnostic associations. Results: Reverse transcription-PCR and Western blot showed that the expression level of CENP-H was higher in NPC cell lines and in immortalized nasopharyngeal epithelial cells than in the normal nasopharyngeal epithelial cell line at both transcriptional and translational levels. By immunohistochemical analysis, we found that 76 of 160 (47.5%) paraffin-embedded archival NPC biopsies showed high expression of CENP-H. Statistical analysis showed that there was a significant difference of CENP-H expression in patients categorized according to clinical stage (P = 0.024) and T classification (P = 0.027). Patients with higher CENP-H expression had shorter overall survival time, whereas patients with lower CENP-H expression had better survival. A prognostic value of CENP-H was also found of the subgroup of N0-N1 tumor classification. Multivariate analysis showed that CENP-H expression was an independent prognostic indicator for patients survival. Conclusions: Our results suggest that CENP-H protein is a valuable marker of NPC progression. High CENP-H expression is associated with poor overall survival in NPC patients.

Prognostic relevance of Bmi-1 expression and autoantibodies in esophageal squamous cell carcinoma

BackgroundOverexpression of Bmi-1 has been observed in a variety of cancers, and it has been suggested to be an independent prognostic marker for the patients. The objective of this study was to determine the level of Bmi-1 expression or its autoantibodies in human esophageal squamous cell carcinoma (ESCC) and to correlate it with clinicopathologic data.MethodsWe first examined Bmi-1 expression in ESCC cell lines and tumor samples by RT-PCR and Western blot analysis. We then analyzed Bmi-1 protein expression in 171 clinicopathologically characterized ESCC cases by immunohistochemistry. In addition, we detected its autoantibodies in sera of patients with ESCC by ELISA.ResultsWe found that Bmi-1 expression was higher in the immortalized cells, cancer cell lines and most cancer tissue than in non-tumorous control tissue at both mRNA and protein level. In addition, Bmi-1 expression was observed in 64.3% (110 of 171) archive ESCC specimen by immunohistochemistry analysis, and the location of Bmi-1 in ESCC was in the nuclei instead of cytoplasm of tumor cells. There was a significant difference of Bmi-1 expression in patients categorized according to stage (P = 0.003) and pN classification (P = 0.047). Multivariate analysis suggested that Bmi-1 expression was an independent prognostic marker for ESCC patients. A prognostic significance of Bmi-1 was also found in the subgroup of T3~T4 and N1 tumor classification. Bmi-1 autoantibodies were detected in sera of 39.0% (62 of 159) ESCC patients. The correlations between anti-Bmi-1 antibodies and tumor stage (P = 0.040), or lymph node status (P < 0.001) were significant.ConclusionsOur results suggest that Bmi-1 protein is a valuable marker of ESCC progression. The presence of Bmi-1 autoantibodies in sera from patients with ESCC may have clinical utility in esophageal cancer diagnosis.

Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells

Epstein–Barr virus (EBV) is implicated as an aetiological factor in B lymphomas and nasopharyngeal carcinoma. The mechanisms of cell-free EBV infection of nasopharyngeal epithelial cells remain elusive. EBV glycoprotein B (gB) is the critical fusion protein for infection of both B and epithelial cells, and determines EBV susceptibility of non-B cells. Here we show that neuropilin 1 (NRP1) directly interacts with EBV gB23–431. Either knockdown of NRP1 or pretreatment of EBV with soluble NRP1 suppresses EBV infection. Upregulation of NRP1 by overexpression or EGF treatment enhances EBV infection. However, NRP2, the homologue of NRP1, impairs EBV infection. EBV enters nasopharyngeal epithelial cells through NRP1-facilitated internalization and fusion, and through macropinocytosis and lipid raft-dependent endocytosis. NRP1 partially mediates EBV-activated EGFR/RAS/ERK signalling, and NRP1-dependent receptor tyrosine kinase (RTK) signalling promotes EBV infection. Taken together, NRP1 is identified as an EBV entry factor that cooperatively activates RTK signalling, which subsequently promotes EBV infection in nasopharyngeal epithelial cells.

Proteomics-based identification of autoantibody against CDC25B as a novel serum marker in esophageal squamous cell carcinoma

This study was aimed to identify tumor proteins that elicit a humoral response in patients with esophageal squamous cell carcinoma (ESCC). Autologous sera of 15 newly diagnosed patients with ESCC and age- and gender-matched 15 healthy controls were analyzed individually for antibody-based reactivity against proteins from 15 homogenized ESCC tissue mixture resolved by two-dimensional PAGE. One protein spot, which reacted with sera from ESCC patients but not with those from controls, was identified to be CDC25B by mass spectrometry and Western blotting. High expression of CDC25B was detected in ESCC cell lines and primary tumor tissues, but not in normal esophageal tissues. In addition, CDC25B expression was significantly higher in tumor tissue of patients with sera positive CDC25B-Abs than that of patients without CDC25B-Abs. Finally, anti-CDC25B antibodies were readily detectable in sera from 45 of 124 (36.29%) patients with ESCC, 13 of 150 (8.67%) patients with other types of cancer and 0 of 102 (0%) of healthy individuals. Thus, CDC25B autoantibodies may have clinical utility in ESCC screening and diagnosis.