Germline Polymorphisms and Length of Survival of Nasopharyngeal Carcinoma: An Exome-Wide Association Study in Multiple Cohorts.

Germline polymorphisms are linked with differential survival outcomes in cancers but are not well studied in nasopharyngeal carcinoma (NPC). Here, a two-phase association study is conducted to discover germline polymorphisms that are associated with the prognosis of NPC. The discovery phase includes two consecutive hospital cohorts of patients with NPC from Southern China. Exome-wide genotypes at 246 173 single nucleotide polymorphisms (SNPs) are determined, followed by survival analysis for each SNP under Cox proportional hazard regression model. Candidate SNP is replicated in another two independent cohorts from Southern China and Singapore. Meta-analysis of all samples (n = 5553) confirms that the presence of rs1131636-T, located in the 3'-UTR of RPA1, confers an inferior overall survival (HR = 1.33, 95% CI = 1.20-1.47, P = 6.31 × 10-8). Bioinformatics and biological assays show that rs1131636 has regulatory effects on upstream RPA1. Functional studies further demonstrate that RPA1 promotes the growth, invasion, migration, and radioresistance of NPC cells. Additionally, miR-1253 is identified as a suppressor for RPA1 expression, likely through regulation of its binding affinity to rs1131636 locus. Collectively, these findings provide a promising biomarker aiding in stratifying patients with poor survival, as well as a potential drug target for NPC.

Orthotopic Xenograft Mouse Model in Esophageal Squamous Cell Carcinoma.

Orthotopic xenograft model recapitulates the faithful organ-specific microenvironment and facilitates analyses involving tumor-stromal interactions that are crucial for developing new-generation cancer therapy. Herein, we describe the detailed rationales and protocols of a versatile orthotopic xenograft model for esophageal squamous cell carcinoma.

Functional characterization of a candidate tumor suppressor gene, Mirror Image Polydactyly 1, in nasopharyngeal carcinoma.

Mirror Image Polydactyly 1 (MIPOL1) is generally associated with congenital anomalies. However, its role in cancer development is poorly understood. Previously, by utilizing the functional complementation approach, microcell-mediated chromosome transfer (MMCT), a tumor suppressor gene, MIPOL1, was identified. MIPOL1 was confirmed to be downregulated in nasopharyngeal carcinoma (NPC) cells and tumor tissues, and re-expression of MIPOL1 induced tumor suppression. The aim of the current study is to further elucidate the functional tumor suppressive role of MIPOL1. In our study, with an expanded sample size of different clinical stages of NPC tumor tissues, we further confirmed the downregulation of MIPOL1 in different cancer stages. MIPOL1 re-expression down-regulated angiogenic factors and reduced phosphorylation of metastasis-associated proteins including AKT, p65, and FAK. In addition, MIPOL1 was confirmed to interact with a tumor suppressor, RhoB, and re-expression of MIPOL1 enhanced RhoB activity. The functional role of MIPOL1 was further validated by utilizing a panel of wild-type (WT) and truncated MIPOL1 expression constructs. The MIPOL1 tumor-suppressive effect can only be observed in the WT MIPOL1-expressing cells. In vitro and nude mice in vivo functional studies further confirmed the critical role of WT MIPOL1 in inhibiting migration, invasion and metastasis in NPC. Overall, our study provides strong evidence about the tumor-suppressive role of MIPOL1 in inhibiting angiogenesis and metastasis in NPC.

Prospective, Multicenter, Phase 2 Trial of Induction Chemotherapy Followed by Bio-Chemoradiotherapy for Locally Advanced Recurrent Nasopharyngeal Carcinoma.

PURPOSE: To evaluate, in a phase 2 study, whether induction docetaxel, cisplatin, and fluorouracil (TPF) followed by weekly docetaxel and cetuximab in concurrence with intensity modulated radiation therapy can improve the treatment outcome for patients with advanced locally recurrent nasopharyngeal carcinoma (rNPC). METHODS AND MATERIALS: Thirty-three patients with rNPC (T3-T4, N0-N1, M0) were recruited. Of these, 19 patients (57.6%) had stage rT3 recurrence, and the rest had stage rT4. Eight patients also had rN1 at the time of relapse. Treatment outcomes and safety were evaluated. RESULTS: Among these 33 patients, 1 died after 1 cycle of TPF, 5 patients withdrew from the study during the induction period because of grade ≥3 toxicities; 27 patients completed the whole course of treatment, but 1 died before any assessment could be made. The median follow-up period was 28.5 months. The progression-free survival and overall survival at 3 years for the whole group were 35.7% and 63.8%, respectively. Among the 26 patients who could be assessed after treatment, the complete response rate was 30.8%, and the locoregional control rate at 3 years was 49.2%. Temporal lobe necrosis (TLN) developed in 8 cases. The rates of grade ≥3 hearing loss, soft tissue necrosis, dysphagia, and trismus were 30.8%, 15.4%, 11.5%, and 19.2%, respectively. Overall, 5 patients died owing to acute (1 after cycle 1 TPF and 1 after completion of bio-chemoradiotherapy) or late (2 epistaxis and 1 TLN) treatment-related complications. CONCLUSIONS: The proposed salvage treatment regimen for advanced locally recurrent NPC could achieve a better treatment outcome than seen in previous studies. However, poor tolerability of induction TPF and the high rate of TLN limit its applicability outside clinical trials.

A CD90(+) tumor-initiating cell population with an aggressive signature and metastatic capacity in esophageal cancer.

Tumor-initiating cells (TIC), also known as cancer stem cells, are regarded widely as a specific subpopulation of cells needed for cancer initiation and progression. TICs have yet to be identified in esophageal tumors that have an increasing incidence in developed countries. Here, we report a CD90(+) cell population found in esophageal squamous cell carcinoma (ESCC), which is endowed with stem cell-like properties and high tumorigenic and metastatic potential. mRNA profiling of these cells suggested pathways through which they drive tumor growth and metastasis, with deregulation of an Ets-1/MMP signaling pathway and epithelial-mesenchymal transition figuring prominently. These cells possessed higher self-renewal activity and were sufficient for tumor growth, differentiation, metastasis, and chemotherapeutic resistance. CD90(+) TICs were isolated and characterized from ESCC clinical specimens as well as ESCC cell lines. In freshly resected clinical specimens, they represented a rare cell population, the levels of which correlated with strong family histories and lymph node metastasis. Our results prompt further study of this CD90(+) population of esophageal TICs as potential therapeutic targets.

Role of STAT3/5 and Bcl-2/xL in 2-methoxyestradiol-induced endoreduplication of nasopharyngeal carcinoma cells.

2-methoxyestradiol (2ME2), an endogenous metabolite of 17-ß-estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 and a poorly differentiated C666-1 cell line. In the present study, we studied the survival factors involved in 2ME2-induced endoreduplicating NPC cells. In the HK-1 cells, knockdown of BcL-xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p-STAT5-Y694), but not p-STAT3 (Y705) and p-STAT3 (S727), in the nucleus of HK-1 cells. Pre-treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl-xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p-STAT3 in the poorly differentiated C666-1 cells. Pharmacological inhibition of STAT3 or Bcl-2/xL resulted in a decrease in endoreduplication of C666-1 cells. Taken together, the expression of p-STAT5 and p-STAT3 was upregulated in 2ME2-induced endoreduplicating HK-1 and C666-1 cells, respectively. Combination of 2ME2 with Bcl-2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.

Role of p38 MAPKs in hypericin photodynamic therapy-induced apoptosis of nasopharyngeal carcinoma cells.

The present study aims to determine the role of mitogen-activated protein kinases (MAPKs) in hypericin-mediated photodynamic therapy (HY-PDT)-induced apoptosis of the HK-1 nasopharyngeal carcinoma (NPC) cells. HY-PDT was found to induce proteolytic cleavage of procaspase-9 and -3 in HK-1 cells. Apoptotic nuclei were observed at 6 h after PDT whereas B-cell leukemia/lymphoma-2-associated-X-protein (Bax) translocation and formation of Bax channel is responsible for the cell death. Increase in phosphorylation of p38 MAPKs and c-Jun N-terminal kinase 1/2 (JNK1/2) was detected at 15-30 min after HY-PDT. The appearance of phosphorylated form of p38 MAPKs and JNK1/2 was inhibited by the singlet oxygen scavenger l-histidine. HY-PDT-induced cell death was enhanced by the chemical inhibitors for p38 MAPKs (SB202190 and SB203580), but not by the JNKs inhibitor SP600125. Knockdown of the p38alpha and p38beta MAPK isoforms by small interfering RNA (siRNA) are more effective than the p38delta in enhancing PDT-induced cell death. Augmentation of apoptosis by p38alpha or p38beta knockdown is also correlated with the increased proteolytic cleavage of procaspase-9 after HY-PDT treatment. Our results suggested that HY-PDT activated p38 MAPKs through the production of singlet oxygen. Inhibition of p38 MAPKs with chemical inhibitors or siRNA enhances HY-PDT-induced apoptosis of the HK-1 NPC cells.

Monochromosome transfer and microarray analysis identify a critical tumor-suppressive region mapping to chromosome 13q14 and THSD1 in esophageal carcinoma.

Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG.

Apoptosis and expression of cytokines triggered by pyropheophorbide-a methyl ester-mediated photodynamic therapy in nasopharyngeal carcinoma cells.

The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5-2µM, MPPa-mediated a drug dose-dependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and beta-chemokines (MIP-1ß, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma.

Tumor suppressive role of a 2.4 Mb 9q33-q34 critical region and DEC1 in esophageal squamous cell carcinoma.

The key genes involved in the development of esophageal squamous cell carcinoma (ESCC) remain to be elucidated. Previous studies indicate extensive genomic alterations occur on chromosome 9 in ESCC. Using a monochromosome transfer approach, this study provides functional evidence and narrows down the critical region (CR) responsible for chromosome 9 tumor suppressing activity to a 2.4 Mb region mapping to 9q33-q34 between markers D9S1798 and D9S61. Interestingly, a high prevalence of allelic loss in this CR is also observed in primary ESCC tumors by microsatellite typing. Allelic loss is found in 30/34 (88%) tumors and the loss of heterozygosity (LOH) frequency ranges from 67 to 86%. Absent to low expression of a 9q32 candidate tumor suppressor gene (TSG), DEC1 (deleted in esophageal cancer 1), is detected in four Asian ESCC cell lines. Stably expressing DEC1 transfectants provide functional evidence for inhibition of tumor growth in nude mice and DEC1 expression is decreased in tumor segregants arising after long-term selection in vivo. There is 74% LOH in the DEC1 region of ESCC primary tumors. This study provides the first functional evidence for the presence of critical tumor suppressive regions on 9q33-q34. DEC1 is a candidate TSG that may be involved in ESCC development.

Monochromosome transfer provides functional evidence for growth-suppressive genes on chromosome 14 in nasopharyngeal carcinoma.

In many cancers, including nasopharyngeal carcinoma (NPC), extensive and multiple regions of allelic loss occur on chromosome 14. However, to date no functionally conclusive tumor suppressor genes have yet been identified on this chromosome. Through use of the monochromosome transfer technique, this study provides functional evidence for the importance of two discrete regions of chromosome 14. A newly established A9 mouse donor cell line containing an intact copy of chromosome 14 was used for transfer of this intact chromosome into the NPC HONE1 cell line. Twelve independently established microcell hybrids demonstrated uniform loss of specific chromosome 14 loci from both endogenous and exogenous alleles. By microsatellite typing and fluorescence in situ hybridization with BAC probes, the two critical regions were localized to 14q11.2-13.1 and 14q32.1. Selective elimination of these regions during hybrid selection was strongly associated with both hybrid survival and tumor growth in vivo. This functional evidence now narrows down the candidate areas for further studies and suggests that at least two hitherto unidentified growth-related genes localized on two critical regions of chromosome arm 14q play an important role in tumorigenesis.