Adjacent Normal Epithelial Cells Research Articles

Transcriptome of sessile serrated adenoma/polyps is associated with MSI-high colorectal cancer and decreased expression of CDX2.

The objective of this study was to elucidate the molecular background of sessile serrated adenoma/polyp (SSA/P) endoscopically resected with comprehensive gene expression analysis. Gene expression profiling was performed for 10 tumor-normal pairs of SSA/P. Cluster analysis, gene set enrichment analysis (GSEA), and consensus molecular subtype (CMS) classification of colorectal cancer (CRC) were applied to our transcriptome analysis. Unsupervised cluster analysis showed that the gene expression profile of SSA/Ps is different from that of adjacent normal epithelial cells, even in the very early stage of tumorigenesis. According to the CMS classification, our microarray data indicated that SSA/Ps were classified as CMS1. GSEA demonstrated a strong association between SSA/P and microsatellite instability-high (MSI-H) CRC (p < 10-5 ). Transcriptome analysis of five MSI-related genes (MSH2, MSH6, MLH1, PMS1, and PMS2) and five CRC-related genes (BRAF, KRAS, APC, TP53, and CDX2) showed that CDX2 expression was most severely decreased in SSA/P. Immunohistochemical staining confirmed that CDX2 protein was reduced compared with the surrounding mucosa. Direct sequencing of the BRAF gene showed that the BRAF V600E mutation was detected in only nine of 36 cases. In a mouse model, BRAF, APC, or CDX2 deficiency indicated that the gene expression pattern with loss of CDX2 is more similar to our SSA/Ps compared with those induced by BRAF or APC mutation. Transcriptome analysis of SSA/Ps showed characteristic gene expression with a strong resemblance to MSI-H CRC. Downregulation of CDX2 expression is an essential molecular mechanism involved in the initial stage of SSA/P tumorigenesis. (UMIN000027365).

The response of phyllodes tumor of the breast to anticancer therapy: An in vitro and ex vivo study.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

Distinct expression patterns of SULT2B1b in human prostate epithelium.

SULT2B1b (sulfotransferase family cytosolic 2B member 1b) catalyzes the sulfate conjugation of substrates such as cholesterol and oxysterols. Our laboratory has previously shown that SULT2B1b inhibition modulates androgen receptor signaling and induces apoptosis in prostate cancer cells. However, the functions of SULT2B1b in the prostate remain poorly understood. We characterized the expression pattern of SULT2B1b in human benign prostate hyperplasia (BPH) as well as prostate cancer to determine the relationship between SULT2B1b and prostate diseases, using immunohistochemistry, immunofluorescence staining, immunoblot, and real-time polymerase chain reaction. SULT2B1b was strongly detected in the prostate epithelium but was absent in the stroma. Significantly lower SULT2B1b was found in primary cancer cells compared with adjacent normal epithelial cells. SULT2B1b further decreased in metastatic cancer cells. Most interestingly, we found, for the first time, that SULT2B1b was much more concentrated in the luminal layer than in the basal layer in both normal prostate and BPH samples. The stronger presence of SULT2B1b in luminal epithelial cells was confirmed by costaining with luminal and basal markers and in sorted paired luminal and basal cells. SULT2B1b expression was induced with prostate organoid differentiation. SULT2B1b inversely correlates with prostate cancer status, with the highest level in the normal epithelium and lowest in the advanced metastatic prostate cancer. Furthermore, SULT2B1b is mostly located within the luminal layer of the prostate epithelium, suggesting that it may be implicated in luminal differentiation.

COP9 signalosome subunit CSN5, but not CSN6, is upregulated in lung adenocarcinoma and predicts poor prognosis.

The COP9 signalosome (CSN) is an evolutionarily conserved complex composed of eight subunits (CSN1-CSN8). Among the CSN subunits, CSN5 and its dimerization partner CSN6 are the only two MPN (Mpr1-Pad1-N-terminal) domain-containing subunits. These two subunits play essential roles in a variety of biological processes, such as cell cycle progression, protein stability and signal transduction. However, their expression patterns and clinical significance in lung cancer are not completely clear. We examined the expressions of both CSN5 and CSN6 in lung adenocarcinoma (LUAD) patients (n=59) using immunohistochemistry analysis, and correlated their expressions with clinicopathological characteristics. MTT cell proliferation assay was performed to determine the effect of CSN5 silencing or overexpression on the growth of lung cancer cells. Knock down or overexpression of CSN5 was confirmed by western blotting. CSN5 expression was elevated in tumor cells, compared to the stromal compartment and adjacent normal epithelial cells. Interestingly, CSN5 was also expressed in the macrophages and lymphocytes adjacent to the tumors. Surprisingly, CSN6 was barely detected in the tumor cells of LUAD patients. Furthermore, we also demonstrated that higher levels of CSN5 were correlated with high tumor-node-metastasis (TNM) stage and worse clinical outcomes. Multivariate Cox regression analysis revealed CSN5 was an independently prognostic factor for LUAD patients. Additionally, in cellular model, depletion of CSN5 expression significantly suppressed the growth of lung cancer cells. COP9 signalosome subunit CSN5, but not CSN6, is upregulated in LUAD. Moreover, CSN5 is a critical regulator for the growth of lung cancer and represents an independent prognostic factor and a promising therapeutic target for LUAD patients.

Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women's breast cancer tumors.

As an experimental model, most studies rely on established human cancer cell lines; however, some genetical or phenotypical differences exist between these cells and their original tumor. Therefore, primary cells isolated directly from tissue are believed to be more biologically relevant tools for studying human and animal biology. Here, we aimed to isolate primary epithelial cancer and normal cells from breast tumors of Iranian women, for the first time. Thus, we isolated the epithelial and fibroblast cells from biopsy samples of patients with breast cancer based on differential centrifugation followed by culture in selective media. Normal epithelial cells obtained from the tissue biopsy away from the core of the tumor, based on the pathological diagnosis. Flow cytometry analysis indicated the positive immunoreactivity of the isolated epithelial cells against CD24 and Epithelial Specific Antigen (ESA/EpCAM), while they displayed a concomitant low expression of CD44 and CD49f. In contrat to fibroblasts, the qPCR data indicated the expression of luminal intracellular cytokeratin (Ck18) in both normal and cancer epithelial cells, but there was no expression of myoepithelial/basal markers, CK5 and vimentin. The epithelialcancer cells were reactive to cytokeratin 19 (CK19) antibody, whereas the normal epithelial cells were not. The expression of calmodulin-like protein (CLP) was also lower in the cancer epithelial cells thanin the normal ones. In conclusion, primary epithelial normal and cancer cells, in addition to the fibroblasts were isolated and characterized from breast tumor of Iranian patients; and CLP expression is suggested as a susceptibility marker for breast cancer screening.

Identification of diagnostic markers in colorectal cancer via integrative epigenomics and genomics data.

Apart from genetic mutations, epigenetic alteration is a common phenomenon that contributes to neoplastic transformation in colorectal cancer. Transcriptional silencing of tumor-suppressor genes without changes in the DNA sequence is explained by the existence of promoter hypermethylation. To test this hypothesis, we integrated the epigenome and transcriptome data from a similar set of colorectal tissue samples. Methylation profiling was performed using the Illumina InfiniumHumanMethylation27 BeadChip on 55 paired cancer and adjacent normal epithelial cells. Fifteen of the 55 paired tissues were used for gene expression profiling using the Affymetrix GeneChip Human Gene 1.0 ST array. Validation was carried out on 150 colorectal tissues using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique. PCA and supervised hierarchical clustering in the two microarray datasets showed good separation between cancer and normal samples. Significant genes from the two analyses were obtained based on a ≥2-fold change and a false discovery rate (FDR) P-value of <0.05. We identified 1,081 differentially hypermethylated CpG sites and 36 hypomethylated CpG sites. We also found 709 upregulated and 699 downregulated genes from the gene expression profiling. A comparison of the two datasets revealed 32 overlapping genes with 27 being hypermethylated with downregulated expression and 4 hypermethylated with upregulated expression. One gene was found to be hypomethylated and downregulated. The most enriched molecular pathway identified was cell adhesion molecules that involved 4 overlapped genes, JAM2, NCAM1, ITGA8 and CNTN1. In the present study, we successfully identified a group of genes that showed methylation and gene expression changes in well-defined colorectal cancer tissues with high purity. The integrated analysis gives additional insight regarding the regulation of colorectal cancer-associated genes and their underlying mechanisms that contribute to colorectal carcinogenesis.

Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells.

Overexpression of NUAK1 is associated with disease-free survival and overall survival in patients with gastric cancer.

Novel (nua) kinase family 1 (NUAK1) is a member of the human adenosine monophosphate-activated protein kinases family, which is overexpressed in multiple human malignancies and thought to be involved in tumor invasion and metastasis ability. Our study is to investigate the association of NUAK1 expression with clinicopathological parameters and prognostic significance of patients with gastric cancer. The expression patterns of the NUAK1 protein in 117 primary archival gastric cancer specimens and 46 adjacent normal epithelial tissues from patients were detected by immunohistochemistry assay. Staining evaluation results were analyzed statistically in relation to various clinicopathological characters, recurrence-free survival and overall survival. High level of NUAK1 expression was detected in gastric cancer, significantly more than in adjacent normal epithelial cells. In gastric cancer, NUAK1 was positively correlated with depth of invasion, lymph node metastasis, pathological stage, surgical resection and histological differentiation. However, no correlations between NUAK1 expression and patients' age, sex, tumor size, location, CA19-9 or CEA were detected. The recurrence-free survival and overall survival were significantly shorter for patients with NUAK1 higher scores than those with NUAK1 lower scores. Multivariate analysis identified NUAK1 was an independent prognostic factor for both recurrence-free survival and overall survival. Our findings provided convincing evidence for NUAK1 overexpression, which was tightly associated with more aggressive tumor behavior and a poor prognosis, indicating that NUAK1 is a valuable molecular biomarker for gastric cancer progression. It might also act as a promising target for both prognostic prediction and therapeutics.

Deletion of the Olfactomedin 4 Gene Is Associated with Progression of Human Prostate Cancer

The olfactomedin 4 (OLFM4) gene is located on chromosome 13q14.3, which frequently is deleted in human prostate cancer. However, direct genetic evidence of OLFM4 gene alteration in human prostate cancer has not yet been obtained. In this study, we investigated the genetics, protein expression, and functions of the OLFM4 gene in human prostate cancer. We found overall 25% deletions within the OLFM4 gene in cancerous epithelial cells compared with adjacent normal epithelial cells that were microdissected from 31 prostate cancer specimens using laser-capture microdissection and genomic DNA sequencing. We found 28% to 45% hemizygous and 15% to 57% homozygous deletions of the OLFM4 gene via fluorescence in situ hybridization analysis from 44 different prostate cancer patient samples. Moreover, homozygous deletion of the OLFM4 gene significantly correlated with advanced prostate cancer. By using immunohistochemical analysis of 162 prostate cancer tissue array samples representing a range of Gleason scores, we found that OLFM4 protein expression correlated inversely with advanced prostate cancer, consistent with the genetic results. We also showed that a truncated mutant of OLFM4 that lacks the olfactomedin domain eliminated suppression of PC-3 prostate cancer cell growth. Together, our findings indicate that OLFM4 is a novel candidate tumor-suppressor gene for chromosome 13q and may shed new light on strategies that could be used for the diagnosis, prognosis, and treatment of prostate cancer patients.

Analysis of Normal-Tumour Tissue Interaction in Tumours: Prediction of Prostate Cancer Features from the Molecular Profile of Adjacent Normal Cells

Statistical modelling, in combination with genome-wide expression profiling techniques, has demonstrated that the molecular state of the tumour is sufficient to infer its pathological state. These studies have been extremely important in diagnostics and have contributed to improving our understanding of tumour biology. However, their importance in in-depth understanding of cancer patho-physiology may be limited since they do not explicitly take into consideration the fundamental role of the tissue microenvironment in specifying tumour physiology. Because of the importance of normal cells in shaping the tissue microenvironment we formulate the hypothesis that molecular components of the profile of normal epithelial cells adjacent the tumour are predictive of tumour physiology. We addressed this hypothesis by developing statistical models that link gene expression profiles representing the molecular state of adjacent normal epithelial cells to tumour features in prostate cancer. Furthermore, network analysis showed that predictive genes are linked to the activity of important secreted factors, which have the potential to influence tumor biology, such as IL1, IGF1, PDGF BB, AGT, and TGFβ.

Matrix metalloproteinase‐9 is associated with disease‐free survival and overall survival in patients with gastric cancer

Matrix metalloproteinase-9 is a member of the Matrix metalloproteinases (MMP) family, which is overexpressed in some solid tumor and thought to enhance the tumor invasion and metastasis ability. Our study is to investigate the association of MMP-9 expression with disease-free survival and overall survival of patients with gastric cancer. Clinical gastric cancer specimens and adjacent normal tissues from 286 patients who had not received neoadjuvant chemotherapy were investigated by immunohistochemistry assay. Staining evaluation results were analyzed statistically in relation to various clinicopathological characters, disease-free survival and overall survival. High level of MMP-9 expression was detected in gastric cancer, significantly more than in adjacent normal epithelial cells. In gastric cancer, MMP-9 was significantly positively correlated with depth of invasion, lymph node metastasis and distant metastasis. However, no correlations between MMP-9 expression and patients' age, sex, tumor location or differentiation status were detected. The disease-free survival and overall survival were significantly shorter for patients with MMP-9 positive than those with MMP-9 negative tumors. Multivariate analysis identified MMP-9 was an independent prognostic factor for both disease-free survival and overall survival. Our findings provided convincing evidence for MMP-9 as an important role in human gastric cancer recurrence and prognosis. It might also serve as a novel target for both prognostic prediction and therapeutics.

Abstract 2684: Telomere length of carcinoma-associated fibroblasts as a biomarker for breast cancer local recurrence

Abstract The use of breast-conservative surgery (BCS) for the treatment of early-stage breast cancers has been increasing and the ability to identify patients at high risk of local recurrence is highly desirable for guiding treatment decisions. Currently, relatively few breast cancer local recurrent risk factors have been identified; those known risk factors, which are based on clinical and histological features of tumor tissue, have limited predictive power. It is known that molecular genetic changes often precede morphologic evidence of malignant transformation. Thus, early molecular genetic alterations in breast tumor and adjacent normal tissues are likely to be more sensitive tools for the identification of patients at increased risk of local recurrence than current histological criteria. A nested case-control study was carried out to evaluate whether telomere length in morphologically normal tissues adjacent to the tumor is predictive of breast cancer local recurrence. One hundred forty-two women who are diagnosed with breast cancer at Lombardi Comprehensive Cancer Center between 1998 and 2005 were included in the study. Cases (N = 42) are patients who had local recurrences of breast cancer after BCS. Controls (N = 100) are patient who had no local recurrence and are matched to cases on year of surgery, age at diagnosis, disease stage and type of surgery. Quantitative telomere fluorescent in situ hybridization (FISH) was used to determine the telomere length of four cell types: cancer cells, carcinoma-associated fibroblast cells (CAFs), adjacent normal epithelial cells and lymphocytes, using 5-micron sections of formalin fixed paraffin-embedded (FFPE) breast tumor tissues. Telomere length of lymphocytes was used to normalize the FISH hybridization variation and relative telomere length (RTL) was defined as telomere length in cells of interest divided by the telomere length of lymphocytes. Multivariate logistic regression was used to assess the association between RTL and breast cancer local recurrence. We found that mean RTL of CAFs was significantly longer in patients with breast cancer local recurrence (mean = 0.99) than in patients without local recurrence (mean = 0.74, p=0.022). When RTL of CAFs were dichotomized using median value of control subjects, patients who had longer RTL in CAFs had 2.4-fold (95% CI=0.86-6.56) increased risk of breast cancer local recurrence than women who had shorter RTL in CAFs. We observed no statistically significant case-control difference in mean RTL of normal epithelial cells adjacent to the tumor (p = 0.712) or in mean RTL of cancer cells (p = 0.285). This study is the first to report that telomere length in tumor stromal cells (CAFs) is associated with breast cancer local recurrence. If confirmed by future studies, telomere length in CAFs has the potential to become a novel molecular tool for the predicting breast cancer local recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2684.

The forkhead box M1 transcription factor as a candidate of target for anti‐cancer immunotherapy

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty‐three human FOXM1‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their ability to induce HLA‐A2‐restricted T cells in HLA‐A2 transgenic mice. FOXM1362‐370 (YLVPIQFPV), FOXM1373‐382 (SLVLQPSVKV), and FOXM1640‐649 (GLMDLSTTPL) peptides primed HLA‐A2‐restricted cytotoxic T lymphocytes (CTLs) in the HLA‐A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA‐A2‐positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA‐A2‐positive FOXM1‐transfectants by peptide‐induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.

The expression of androgen‐responsive genes is Up‐Regulated in the epithelia of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is one of the most common diseases among aging men in the United States. In addition to aging, the presence of androgens is another major risk factor in BPH development. However, whether androgen signaling is altered in BPH remains unclear. To determine androgen signaling in BPH, we characterized the expression of four different androgen-responsive genes, Eaf2/U19, ELL2, FKBP5, and PSA, in BPH and adjacent normal glandular epithelial cells. A set of 17 BPH specimens were resected from patients over 60 years of age with clinical symptoms of BPH. Laser-capture microdissection (LCM) was used to isolate glandular epithelial cells from BPH areas and adjacent normal areas, separately. LCM isolated cells from individual specimens were lysed and RNA isolation, reverse transcription, and real-time PCR were performed using CellsDirect One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA). All of the assayed genes displayed increased expression, from approximately 2- to approximately 6-fold, in BPH as compared to the adjacent normal epithelial cells. We also generated a composite androgen response index based on the expression levels of the four genes, which provides a reliable readout for overall androgen action. Our study showed that the composite androgen response index in BPH is approximately 4-fold as compared to that in the adjacent normal tissues. Androgen signaling is significantly elevated in BPH relative to the adjacent normal prostate. Understanding the mechanisms causing elevated androgen signaling may lead to novel approaches for prevention and/or treatment of BPH.

New cytotoxic rosamine derivatives selectively accumulate in the mitochondria of cancer cells

Conventional cytotoxic anticancer drugs that target all rapidly dividing cells are nonselective in their mechanism of action, because they disrupt essential components that are crucial to both malignant and proliferating normal cells. Instead, targeting cellular functions that are distinctly different between normal and cancer cells may provide a basis for selective killing of tumor cells. One such strategy that is still largely unexplored is to utilize the relatively higher negative mitochondrial membrane potential in carcinoma cells compared with adjacent normal epithelial cells to enhance accumulation and retention of cytotoxic lipophilic cations in the former. In this study, the anticancer activities of a new class of rosamines with cyclic amine substituents and their structure-activity relationships were investigated. From an in-vitro cell growth inhibition assay, 14 of the rosamines inhibited the growth of human leukemia HL-60 cells by 50% at micromolar or lower concentrations. Derivatives containing hydrophilic substituents had less potent activity, whereas aryl substitution at the meso position conferred extra activity with thiofuran and para-iodo aryl substitutions being the most potent. In addition, both compounds were at least 10-fold more cytotoxic than rhodamine 123 against a panel of cell lines of different tissue origin and similar to rhodamine 123, exhibited more cytotoxicity against cancer cells compared with immortalized normal epithelial cells of the same organ type. In subsequent experiments, the para-iodo aryl substituted rosamine was found to localize exclusively within the mitochondria and induced apoptosis as the major mode of cell death. Our results suggest that these compounds offer potential for the design of mitochondria-targeting agents that either directly kill or deliver cytotoxic drugs to selectively kill cancer cells.

Telomere Attrition in Cancer Cells and Telomere Length in Tumor Stroma Cells Predict Chromosome Instability in Esophageal Squamous Cell Carcinoma: A Genome-Wide Analysis

Previous studies showed that chromosomal instability was common in esophageal squamous cell carcinoma (ESCC); however, the mechanisms underlying this instability are unknown. Individuals with deficiencies in telomere maintenance are susceptible to enhanced telomere loss during cell proliferation; such deficiencies could result in telomere dysfunction and genomic instability. We investigated the association between genome-wide chromosomal changes in cancer cells and telomere length/attrition in cancer/stroma cells in 47 ESCC patients. Genome-wide detection of loss of heterozygosity was performed using the Affymetrix GeneChip single nucleotide polymorphism arrays. Telomere length was assessed separately for cancer cells, carcinoma-associated fibroblasts (CAF), infiltrative lymphocytes, and adjacent normal epithelial cells by quantitative fluorescent in situ hybridization using paraffin-embedded sections. Telomere length differed significantly among cell types, such that length in infiltrative lymphocytes > CAFs > cancer cells. Shortened telomeres were observed in cancer cells in 44 of 47 (94%) of the tumors examined. Telomere length in CAFs was significantly associated with chromosomal instability on 4q and 13q and lymphocyte telomere length was significantly associated with instability on chromosomal arms 15q. Although telomere length in cancer cells was not associated with chromosome arm instability, telomere attrition in cancer cells, defined as the telomere length in CAFs minus the telomere length in cancer cells, was significantly associated with chromosomal instability on 13q and 15q. This study provides evidence that telomere shortening is a common genetic alteration in ESCC and that chromosome arm instability is related to both telomere attrition in cancer cells and telomere length in tumor stroma cells.

Proteomic Comparison of Nasopharyngeal Cancer Cell Lines C666-1 and NP69 Identifies Down-Regulation of Annexin II and β2-Tubulin for Nasopharyngeal Carcinoma

AbstractContext.—Nasopharyngeal carcinoma (NPC), common in southern China and North Africa, has a complex etiology involving interplay between viral, environmental, and hereditary factors and is almost constantly associated with the Epstein-Barr virus. Since the prognosis of locally advanced and metastatic diseases is poor, increased understanding of the pathogenesis of NPC would be important for discovering novel markers for patients' management.Objectives.—To compare the proteomic expression profile between an Epstein-Barr virus–associated NPC cell line (C666-1) and a normal NP cell line (NP69). The proteins with differential expression were analyzed in 40 undifferentiated NPC paraffin-embedded specimens.Design.—Differentially expressed proteins discovered between the two cell lines were identified by mass spectrometry. After confirmation by immunocytochemical staining, their expression in patient samples was measured using 40 pairs of undifferentiated NPCs together with their adjacent normal epithelia.Results.—Proteomic findings indicated that adenosine triphosphate synthase α chain was up-regulated, whereas annexin II, annexin V, β2-tubulin, and profilin 1 were down-regulated. After confirming the results in agar-processed cell lines, annexin II and β2-tubulin expression were found to be lower in tumor cells than in adjacent normal epithelial cells in 100% and 90% of the patients' specimens, respectively. Finally, annexin II down-regulation was positively associated with lymph node metastasis, suggesting that it may be a prognostic factor in NPC.Conclusions.—The results suggest that annexin II and β2-tubulin down-regulation is important in NPC formation and may represent potential targets for further investigations.

Proteomic analysis of global alteration of protein expression in squamous cell carcinoma of the esophagus

Squamous cell carcinoma of the esophagus (ESCC), a major subtype of esophageal carcinoma, is one of the aggressive cancers with worst prognosis in the world. The dismal outcome of ESCC is attributed to multiple reasons including its aggressive nature, largely unknown molecular mechanism of its progression, and the lack of biomarkers for early detection and effective prediction of its clinical behavior. To identify proteins with prognostic and/or predictive value, we applied a proteomics strategy to quantify proteins differentially expressed in ESCC using matched samples of carcinoma and adjacent normal epithelial cells. The analysis led to identification of 28 proteins aberrantly expressed in cancer cells with changes of at least three-fold in ESCC relative to normal squamous epithelial cells. These changes represent functional alterations of essential proteins for normal cellular physiology, accounting for many cellular changes involved in development of ESCC, including cell transformation, loss of differentiation, tumor growth, apoptosis, tumor invasion, and cell metabolism. The differentially expressed proteins shed new insights on the mechanism of tumorigenesis and provide candidate biomarkers for early detection of ESCC.

Epstein-Barr Virus and CD21 Expression in Gastrointestinal Tumors

Epstein-Barr virus (EBV) was found in 7-17% of gastric adenocarcinomas, including lymphoepithelioma-like carcinomas, although its significance has not been clear. In addition, 20-30% of malignant lymphomas arising in the gastrointestinal tract have been known to express the EBV genome. Several lines of evidence indicate that EBV has been shown to infect both B lymphocytes and squamous epithelial cells via CD21 molecule in vivo and in vitro. The expression of CD21 in EBV-associated gastrointestinal tumors, however, has remained controversial. To determine the presence of CD21, an EBV receptor, in the EBV-associated gastrointestinal tumors, we, first, examined the EBV genome in sixty seven patients with either gastrointestinal adenocarcinomas or malignant lymphomas using in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs) and PCR for EBNA-1. Then, the investigation of CD21 expression was performed only in the EBV-positive tumors with immunohistochemical method for CD21 antigen on paraffin sections. EBERs were detected in 6 out of 26 gastric adenocarcinomas, 2 of 24 colonic adenocarcinomas, and 8 of 17 malignant lymphomas. EBERs were more prevalent in the malignant lymphomas originating from the small and large intestine (6/6) than from the stomach (2/11), and were detected in both B and T cell phenotypes. EBNA-1 was amplified in 11 of 16 EBERs-positive cases. Interestingly, however, none of the EBV-positive six gastric adenocarcinomas and eight malignant lymphomas expressed the CD21 on the cell surfaces or cytoplasm of both tumor cells and adjacent normal epithelial cells. These results suggest that EBV infection in the gastrointestinal malignancies would be mediated via different routes besides the CD21 or a new receptor distinct from CD21.