Epigenetic Inactivation of <i>Inositol polyphosphate 4-phosphatase B</i> (<i>INPP4B</i>), a Regulator of PI3K/AKT Signaling Pathway in EBV-Associated Nasopharyngeal Carcinoma

<div><p>Nasopharyngeal carcinoma (NPC) is a common viral-associated neoplasm in which multiple signaling cascades are interfered with by Epstein-Bar virus (EBV) latent proteins and various genetic alterations. Aside from the previously reported <i>PIK3CA</i> amplification, we examined the role of INPP4B, a negative regulator of the PI3K/AKT signaling pathway in the development of NPC. By RT-PCR and Western blotting, we revealed that the expression of <i>INPP4B</i> was down-regulated in all five established EBV-positive tumor lines. While <i>INPP4B</i> was consistently expressed in normal nasopharyngeal epithelial cells, downregulation of <i>INPP4B</i> was found in 32/65 (49.2%) of primary tumors by immunohistochemistry. Furthermore, our study also demonstrated the hypermethylation of the 5′CpG island of <i>INPP4B</i> in the tumors in which <i>INPP4B</i> transcription was downregulated. Notably, the re-expression of <i>INPP4B</i> was detected in the NPC cells treated with the demethylation agent (5-aza-2′deoxycytidine). Our study showed that promoter hypermethylation was the major mechanism for transcriptional silencing of <i>INPP4B</i> in NPC. Furthermore, restoration of INPP4B expression significantly suppressed PI3K/AKT downstream signals in the NPC C666-1 cells. <i>In vivo g</i>rowth inhibition was clearly demonstrated in the tumor cells stably expressing INPP4B. The findings indicate that epigenetic inactivation of <i>INPP4B</i> is one of the key mechanisms in activating PI3K/AKT signaling cascade and playing a role in the tumorigenesis of NPC.</p></div

Activation of the PI3K/AKT pathway in EBV+ve NPC tumor cell line and xengrafts.

<p>(A) By Western blotting, p-AKT (Thr308) was detected in all EBV-positive NPC tumor lines and p-AKT (Ser473) was found in C666-1, C15 and xeno-99186. The weak expression of p-AKT was shown in the NP69 cells. No AKT activation was observed in the EBV-ve NPC cell line HK-1 and the breast cancer cell line MCF7. p-mTOR was found in all of the cell lines except C17 and xeno-1915 which also shown absence of mTOR expression. The phosphorylation of GSK-3β (Ser9) was detected in xeno-99186 and C17. A high level of PTEN expression was found in the EBV+ve NPC tumor lines. Relative protein expression was calculated using densitometry with C666-1 at 1. The ratios of the phosphorylation and total protein of AKT, mTOR and GSK-3β were also indicated. (B) Suppression of AKT and mTOR phosphorylation was detected in NPC C666-1 cells transient transfected with <i>INPP4B</i>. Relative protein expression was calculated using densitometry with vector control (24hr) at 1. The ratios of the phosphorylation and total protein of AKT and mTOR were indicated.</p

Expression of INPP4B in NPC. (A) RT-PCR analysis of <i>INPP4B</i> transcription in NPC, gastric and cervical cancer cell lines.

<p>The reduced expression of <i>INPP4B</i> was detected in the EBV+ve NPC tumor lines when compared with the EBV-negative NPC cell line HK1 and immortalized nasopharyngeal epithelial cells NP69. Loss of <i>INPP4B</i> transcription was also detected in a gastric cancer cell line NKM28 and a cervical cancer cell line HeLa. The RT-PCR experiments were performed in duplicate. (B) The reduced INPP4B protein expression of EBV-positive NPC tumor lines was detected by Western blotting. A high level of INPP4B expression was found in the EBV-negative NPC cell line HK1. The experiment were carried out in duplicate (C) By IHC staining, a reduction in, or loss of, INPP4B expression was detected in the NPC tumor T3, T5 and T10. In tumor T3, intensive staining of INPP4B was demonstrated in the normal nasopharyngeal epithelial cells (red arrow). Representative NPC cases with medium (T11 and T15) and high expression levels of INPP4B (T6) are shown. NPC tumor cells are indicated (*).</p

Characteristics of the EBV-positive NPC Patients (n = 65).

<p>Characteristics of the EBV-positive NPC Patients (n = 65).</p

L'Ordinamento sportivo: caratteri generali Principali testi normativi

We present a new method, OMSV, for accurately and comprehensively identifying structural variations (SVs) from optical maps. OMSV detects both homozygous and heterozygous SVs, SVs of various types and sizes, and SVs with or without creating or destroying restriction sites. We show that OMSV has high sensitivity and specificity, with clear performance gains over the latest method. Applying OMSV to a human cell line, we identified hundreds of SVs &gt;2 kbp, with 68 % of them missed by sequencing-based callers. Independent experimental validation confirmed the high accuracy of these SVs. The OMSV software is available at http://yiplab.cse.cuhk.edu.hk/omsv/

CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

<div><p>Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by <em>in vitro</em> and <em>in vivo</em> assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. <em>FOXN4</em>, <em>GLI1</em>), immune response (<em>CCR7</em>, <em>IL8</em>) and transmembrane transport (e.g. <em>ABCC3</em>, <em>ABCC11</em>) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 <em>in vitro</em>. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.</p> </div