Epithelial Specimens Research Articles

Cytokine tumor necrosis factor alpha induces intestinal epithelial barrier dysfunction

Background and aimsEpithelial barrier dysfunction is involved in a number of diseases in the body. The mechanism is to be further understood. The present study aimed to investigate the role of one of the common microbial products, flagellin (FGN), in the induction of intestinal epithelial barrier dysfunction. MethodsWe collected the colon epithelium specimens from 40 patients with ulcerative colitis (UC), 40 patients with Crohn’s disease (CD) and 40 healthy volunteers. The expression of toll like receptors (TLR)5 of the specimens was assessed by RT-PCR and western blotting. The expression of tumor necrosis factor alpha (TNFα) and its role in compromising the barrier function in the intestinal epithelial cells, T84 cells, were observed by a cell culture model. ResultsThe results showed that the expression of TLR5 was observed in the colon epithelium of healthy subjects that was increased in UC patients and further increased in CD patients. Treating T84 cells with FGN increased the expression of TNFα in the cells that caused the T84 cell apoptosis as well as compromised the T84 monolayer barrier function, which could be prevented by knocking down the gene of TNFα in T84 cells. ConclusionsWe conclude that the human colon epithelial cells express detectable TLR5 that is increased in patients with CD and UC. The exposure to FGN can increase the expression of TNFα that further compromises the intestinal epithelial barrier function.

Pathological implication and function of Bcl2-inhibitor of transcription in ovarian serous papillary adenocarcinomas

The Bit-1 protein appears to be a part of the integrin-specific signaling pathway involved into anoikis. When Bit1 is released from the mitochondria into the cytoplasm it can elicit caspase-independent apoptosis. The expression of Bit1 in 78 serous papillary adenocarcinomas and 78 normal epithelial ovarian tissue specimens was analyzed by immunohistochemistry. We also investigate Bit1 function by transfection. Bit1 was expressed in 100% and 33.3% of ovarian cancers and normal epithelial tissues, respectively, and its expression was significantly correlated with histologic grade and overall survival. However, Bit1 expression was not associated with age. We also confirmed that Bit1 overexpression in cytosol of Caov-3 cells induced apoptosis. Bit1 may be a useful pathological marker and a prognostic marker for serous papillary adenocarcinomas outcome. Its pro-apoptotic property also makes it a potential gene medicine for ovarian cancers therapy.

Expression of semaphorin 3A and neuropilin 1 with clinicopathological features and survival in human tongue cancer

Objective: To investigate the association between semaphorin 3A (SEMA 3A) and its receptor neuropilin 1 (NRP1) and the clinicopathologic characteristics of patients with tongue cancer. Study Design: Forty-three tongue squamous cell carcinoma specimens were included. Immunohistochemical staining of SEMA3A and NRP1 was performed on 15 normal tongue epithelium specimens and the 43 tumour specimens. Immunoreactivity was evaluated based on the staining intensity and distribution score. Statistical analyses were performed using Chi-squared and Spearman tests and Kaplan-Meier analysis. Results: SEMA3A was significantly down-regulated in tongue cancer compared with normal tongue (P=0.025), while NRP1 was over-expressed in tumours (P<0.001). SEMA3A expression inversely correlated with nodal metastasis (P=0.017). NRP1 expression did not correlate with any clinicopathological characteristics. Higher SEMA3A expression strongly predicted longer survival (P=0.005). Scores for the NRP1/SEMA3A ratio of ≥1 predicted shorter survival (P=0.045). Conclusions: Aberrant expression of SEMA3A and its receptor NRP1 might be involved in the development of tongue cancer and might be useful prognostic markers in this tumour type. Key words:Semaphorin 3A, neuropilin 1, tongue, squamous cell carcinoma.

Upregulation of β2-microglobulin expression in progressive human oral squamous cell carcinoma

The aim of the present study was to investigate β2-microglobulin (β2-M) expression in normal oral mucosa and progressive oral squamous cell carcinoma (OSCC) and to assess the clinical significance of β2-microglobulin expression. The study included 10 cases of normal oral mucosa epithelium specimens, 55 cases of primary OSCC specimens, and 25 cases of OSCC metastasis specimens. Immunohistochemistry was used to determine β2-M expression, and its correlation with clinicopathological factors in progressive OSCC was evaluated. Immunohistochemistry showed that strong β2-M expression was significantly asscociated with tumor size (T3, T4 vs. T1, T2; P=0.001), positive node status (N positive vs. N negative; P=0.000) and advanced clinical stage (III, IV vs. I, II, P=0.000) in primary OSCC lesions. Compared to primary OSCC lesions, the frequency of β2-M expression was significantly increased in metastatic OSCC lesions (P=0.02). In addition, in vitro results from Western blotting showed increased β2-M expression in the two OSCC lines studied. Therefore, we speculate that the up-regulation of β2-M expression may contribute to the oncogenesis of human oral mucosa, tumor invasion and metastasis.

Protein Expression Signatures for Inhibition of Epidermal Growth Factor Receptor-mediated Signaling

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells, and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple reaction monitoring analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier's disease who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, Jagged-1, and Claudin 4, were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically useful EGFR inhibition signature, the results confirm the hypothesis that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical approach to derive and test protein expression signatures for drug action on signaling networks.

Aberrant methylation of secreted protein, acidic and rich in cysteine in human laryngeal and hypopharyngeal carcinoma

Secreted protein, acidic and rich in cysteine (SPARC) has been found to be involved in various stages of tumor progression such as migration, invasion, extracellular matrix deposition and angiogenesis. To obtain an insight into the role of SPARC in the progression of laryngeal and hypopharyngeal carcinoma, we investigated SPARC transcription levels and promoter methylation in carcinoma cell lines and primary tumors. Reverse transcription-PCR showed that SPARC was silenced in laryngeal and hypopharyngeal carcinoma cell lines, in which aberrant promoter methylation was detected. Hypermethylation of SPARC was detected in 56.1% (23/41) of laryngeal carcinoma and 70.0% (7/10) of hypopharyngeal carcinoma biopsies, but only in 11.1% (1/9) of normal epithelial specimens by a methylation-specific PCR assay. Bisulphite genomic sequencing indicated that CpG sites in the SPARC promoter were heavily methylated in cell lines and primary tumors. Moreover, pharmacological demethylation treatment rescued SPARC expression with 5-aza-2'-deoxycytidine (5-aza-dC) in the laryngeal carcinoma cell lines. SPARC promoter hypermethylation was significantly correlated with lymph node metastasis (p<0.01). Our findings suggest that hypermethylation of SPARC is a frequent and tumor-specific event in laryngeal and hypopharyngeal carcinomas and may serve as a biomolecular marker for diagnosis and prognosis.

Abstract 2203: Implication of GPRC5A loss in lung carcinogenesis in patients with and without chronic obstructive pulmonary disease

Abstract Lung cancer is often associated with inflammation induced by cigarette smoke. In addition, chronic obstructive pulmonary disease (COPD), typically associated with inflammation, is a leading cause of morbidity and mortality in the United States and presents an increased risk of lung cancer development compared to patients without COPD. Mice with knockout of both alleles of the G-protein coupled receptor, family C, group 5, member A (Gprc5a) gene develop spontaneous lung adenomas and adenocarcinomas at a much higher incidence than their wild-type littermates, indicating that this gene is a novel lung-specific tumor suppressor gene. Interestingly, the majority of tumors in the Gprc5a knockout mice are associated with inflammatory cell infiltration, possibly due to increased NF-κB activation in mouse lung epithelial cells and tissues. Furthermore, the human GPRC5A can suppress NF-κB activation in human lung adenocarcinoma cells. Therefore, in the present study we investigated the expression patterns of GPRC5A in clinical specimens, including normal bronchial epithelia (NBE) of COPD patients with and without overt lung cancer as well as in non-small cell lung cancer (NSCLC) tumors by immunohistochemical (IHC) analysis. We performed IHC analysis of a tissue microarray (TMA) comprised of 311 lung adenocarcinomas and 166 squamous cell carcinomas (SCCs), as well as of normal bronchial epithelial specimens from 50 patients with COPD, which included 24 cancer-free cases and 26 cases with NSCLC (12, adenocarcinoma; 12, SCC; 2, bronchioalveolar carcinoma). Kruskal-Wallis test was used to compare GPRC5A levels among histology levels. All statistical tests were two-sided, and p values of 0.05 or less were considered to be statistically significant. Cytoplasmic GPRC5A expression was significantly higher in lung adenocarcinomas compared to SCCs (p&amp;lt;0.001). Moreover, GPRC5A expression exhibited a positive correlation with never-smoking status (p=0.005). Interestingly, we noted a statistically significant inverse correlation between the expression of GPRC5A and that of NF-κB (p&amp;lt;0.001), which we had previously found to be activated and elevated following loss of the GPRC5A tumor suppressor. Furthermore, analysis of two NBE obtained from each of 50 COPD patients demonstrated statistically significant decreased expression of GPRC5A in NBE of COPD patients with NSCLC compared to NBE from NSCLC-free COPD patients (p&amp;lt;0.001). Our findings demonstrate that decreased GPRC5A expression may be associated with development of lung malignancies, especially in individuals with chronic lung inflammation, and pinpoints GPRC5A's potential suppressive effects on a lung tumor-promoting microenvironment. Assessment of GPRC5A's potential use as a risk factor for NSCLC development in COPD patients is warranted. Supported by the Samuel Waxman Cancer Research Foundation and by W81XWH-04-1-0142. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2203. doi:10.1158/1538-7445.AM2011-2203

Roles of Krüppel-like factor 4 in oesophageal epithelial cells in Barrett's epithelium development

ObjectivesThe mechanism of transformation to intestinal metaplasia in Barrett's oesophagus has not been clarified. We previously reported that bile acids activate the Cdx2 promoter via nuclear factor kappa B (NF-κB)...

Novel Surgical Approaches for Sampling the Ovarian Surface Epithelium and Proximal Fluid Proteome

The pathogenesis of ovarian, fallopian tube, and peritoneal cancers has been difficult to elucidate despite intense effort. Recently, though, the care of women felt to be at high risk due to a strong family history of breast and/or ovarian cancer or a known germline BRCA1 or BRCA2 mutation has provided potential insight into the development of these malignancies. Risk-reducing surgical removal of the fallopian tubes and ovaries, called risk-reducing bilateral salpingo-oopherectomy (RRBSO), is commonly performed as a laparoscopic procedure to minimize recovery time. We describe here an optimized surgical sampling workflow for analyzing the proteomes of peritoneal, fallopian tube, and ovarian surface epithelial (OSE) specimens collected at the time of laparoscopic RRBSO, a technique which has not been described previously. This methodology presents a unique opportunity for closer examination of the proteomic alterations in the tissues at risk for malignant transformation in women with an inherited susceptibility to ovarian, fallopian tube, and peritoneal cancer development.

Proliferation of cultured lens epithelial cells and association with possible risk factors for posterior capsular opacification

Abstract Purpose To determine the proliferative capacity of human lens epithelial cells (HLEC) in culture and investigate possible associations with clinical characteristics of the donors, such as age, gender, pseudoexfoliations, uveitis and diabetes. Methods Pieces of lens capsule and adhering lens epithelial cells were obtained through capsulorhexis at routine cataract surgery using phacoemulsification technique, with informed consent from the patients. These human lens epithelium specimens were cultured in a humidified CO2 incubator using standard culture medium and 5% fetal calf serum for two weeks. After one and two weeks respectively, cultured cells were stained with carboxy‐fluorescein diacetate succinimidyl ester (CFDA SE), after which image processing software was used to determine the area of the confluent epithelial cell layer in relation to the size of the original capsule specimen. Results The increase in the area of confluent HLEC showed a negative correlation with age at the second week of growth and with diabetes at the first week after surgery. Female gender was associated with higher rate of cell proliferation when considering the whole culture period. The presence of pseudoexfoliations in vivo or a history of uveitis did not significantly affect cell growth in culture postoperatively. Nor did installation of xylocain, trypan blue or phenylephrine in the anterior chamber during surgery affect cell proliferation. Conclusion High age, male gender and diabetes are associated with lower rate of proliferation of lens epithelial cells in culture and may hence be protective against posterior capsular opacification.

Long-term histological examination of inferior concha after radiofrequency thermal ablation

To examine the histological effects of radiofrequency thermal ablation on the inferior concha epithelium and subepithelium, over five years post-treatment. Inferior nasal concha epithelial biopsy specimens were examined histologically before and four, 30, 48 and 60 months after radiofrequency treatment, in six patients with inferior nasal concha hypertrophy. At four months post-treatment, there was proliferation of blood vessels, increased inflammatory cells and a slightly decreased number of glands. At 30 months post-treatment, the number of inflammatory cells and glands had decreased, but signs of increased vascular proliferation, fibrosis and granulation were seen. At 48 and 60 months post-treatment, the number of inflammatory cells and blood vessels had decreased significantly, the number of glands had increased, and lobulation was observed. Radiofrequency thermal ablation does not cause carbonisation or osteitis in the inferior concha. The resultant fibrosis causes contraction of the concha and only minor tissue destruction (as shown by the persistence of submucosal glands).

Experimental study of cornea reparation after epipolis laser in situ keratomileusis

目的 探讨微型角膜刀法准分子激光上皮下角膜磨镶术(Epi-LASIK)术后角膜的愈合规律,并与准分子激光屈光性角膜切削术(PRK)比较.方法 建立兔近视性Epi-LASIK及近视性改良PRK动物模型,用锥虫蓝-茜素红活性染色法观察Epi-LASIK术后角膜上皮细胞活性,裂隙灯显微镜下观察角膜瓣变化、上皮修复过程和上皮下雾状混浊(haze)形成情况,通过角膜组织病理学观察角膜上皮及基质的愈合反应.结果 Epi-LASIK术后角膜上皮愈合时间为(4.67±0.41)d,而PRK术后为(2.75±0.27)d,差异有统计学意义(t=9.550,P=0.000).PRK术后即刻和3d、5d角膜上皮瓣细胞活性率分别为(85.83±2.07)%、(48.67±3.41)%、(91.33±3.10)%,差异有统计学意义 (F=215.060,P=0.000).Epi-LASIK术后角膜瓣逐渐融解并被新生上皮取代,上皮愈合过程较改良PRK缓慢.PRK术后haze分级高于Epi-LASIK,差异有统计学意义(Z=2.27,P=0.02).角膜组织病理学检查显示,术后1个月时2组上皮均明显增厚,3个月时上皮细胞排列仍不规则,6个月时上皮细胞形态基本恢复正常,术后3～6个月时Epi-LASIK组角膜上皮厚度已基本接近正常,与PRK组比较差异有统计学意义(P＜0.05).术后3～6个月时Epi-LASIK组角膜前基质细胞数明显低于PRK组(P＜0.05).结论 Epi-LASIK术后上皮瓣活性随时间的推移而降低,上皮修复较慢.与改良PRK比较,Epi-LASIK术后上皮细胞形态变化和上皮增厚程度较轻,角膜基质细胞恢复快,haze程度明显减轻。

Functional Role and Prognostic Significance of CD157 in Ovarian Carcinoma

CD157, an ADP-ribosyl cyclase-related cell surface molecule, regulates leukocyte diapedesis during inflammation. Because CD157 is expressed in mesothelial cells and diapedesis resembles tumor cell migration, we investigated the role of CD157 in ovarian carcinoma. We assayed surgically obtained ovarian cancer and mesothelial cells and both native and engineered ovarian cancer cell lines for CD157 expression using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), and for adhesion to extracellular matrices, migration, and invasion using cell-based assays. We investigated invasion of human peritoneal mesothelial cells by serous ovarian cancer cells with a three-dimensional coculture model. Experiments were performed with or without CD157-blocking antibodies. CD157 expression in tissue sections from ovarian cancer patients (n = 88) was examined by immunohistochemistry, quantified by histological score (H score), and categorized as at or above or below the median value of 60, and compared with clinical parameters. Statistical tests were two-sided. CD157 was expressed by ovarian cancer cells and mesothelium, and it potentiated the adhesion, migration, and invasion of serous ovarian cancer cells through different extracellular matrices. CD157-transfected ovarian cancer cells migrated twice as much as CD157-negative control cells (P = .001). Blockage of CD157 inhibited mesothelial invasion by serous ovarian cancer cells in a three-dimensional model. CD157 was expressed in 82 (93%) of the 88 epithelial ovarian cancer tissue specimens. In serous ovarian cancer, patients with CD157 H scores of 60 or greater had statistically significantly shorter disease-free survival and overall survival than patients with lower CD157 H scores (CD157 H score > or =60 vs <60: median disease-free survival = 18 months, 95% confidence interval [CI] = 5.92 to 30.07 vs unreached, P = .005; CD157 H score > or =60 vs <60: median overall survival = 45 months, 95% CI = 21.21 to 68.79 vs unreached, P = .024). Multivariable Cox regression showed that CD157 is an independent prognostic factor for recurrence (hazard ratio of disease recurrence = 3.01, 95% CI = 1.35 to 6.70, P = .007) and survival (hazard ratio of survival = 3.44, 95% CI = 1.27 to 9.31, P = .015). CD157 plays a pivotal role in the control of ovarian cancer cell migration and peritoneal invasion, and it may be clinically useful as a prognostic tool and therapeutic target.

CpG hypermethylation of human four-and-a-half LIM domains 1 contributes to migration and invasion activity of human bladder cancer

We previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacologic demethylating agent (5-aza-dC). We focused on the human four-and-a-half LIM domains 1 (FHL1) gene which was selected on the basis of previous microarray data analysis. Because LIM domains provide protein-protein binding interfaces, FHL genes play an important role in cellular events, such as focal adhesion and differentiation, by interacting with the target protein as either a repressor or activator. We hypothesized that inactivation of the FHL1 gene through CpG methylation contributes to cell viability including migration and invasion activity of human BC. After 5-aza-dC treatment, the expression levels of FHL1 mRNA transcript markedly increased in all cell lines tested, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR). The methylation index of FHL1 in our samples was significantly higher in 70 BC specimens than in 10 normal bladder epithelium (NBE) specimens (63.9+/-25.5 and 0.3+/-0.2, respectively; p=0.0066). Conversely, FHL1 mRNA expression was significantly lower in the BC specimens than in the NBE ones (0.331+/-0.12 and 2.498+/-0.61, respectively; p=0.0011). In addition, significant inhibitions of wound healing (45.78+/-6.2, and 100+/-0, respectively; p=0.009) and of cell invasion (18.5+/-2.3 and 95.2+/-2.4, respectively; p=0.02) were observed in stable FHL1-transfected cells than in the control BC cells. In conclusion, we found that the mechanism of FHL1 down-regulation in BC is through CpG hypermethylation of the promoter region. FHL1 gene inactivation by CpG hypermethylation may thus contribute to migration and invasion activity of BC.

Distinct Expression Levels and Patterns of Stem Cell Marker, Aldehyde Dehydrogenase Isoform 1 (ALDH1), in Human Epithelial Cancers

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDHbr tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.

Minichromosome maintenance‐7 and geminin are reliable prognostic markers in patients with oral squamous cell carcinoma: immunohistochemical study

Minichromosome maintenance (MCM) proteins act as the origin licensing machinery that regulates initiation of DNA replication. Geminin is a licensing repressor and prevents reinitiation of DNA replication by blocking reloading of MCM proteins at replication origins. Recent studies have proposed that MCM7 and geminin may be sensitive proliferative and prognostic markers of various malignant tumors. This study aimed to analyze the expression of MCM7 and geminin to clarify pathobiological significance in human oral squamous cell carcinomas (OSCCs). We performed immunohistochemical analysis on 10 specimens of normal oral epithelia, 50 lesions with dysplasia and 113 OSCCs. Labeling indices (LIs) for MCM7, geminin and Ki-67 were evaluated, comparing with clinicopathological profiles. The mean LIs for MCM7 were 29.2% for normal epithelia, 32.2% for dysplasias, and 51.1% for OSCCs; the value was significantly higher in the last than in the former two (P < 0.01). The mean LIs for geminin were 6.8% for normal epithelia, 9.2% for dysplasias, and 21.3% for OSCCs; the value was significantly higher in the OSCCs (P < 0.01). The MCM7 LIs were correlated with the histological grade of OSCCs, in which the highest LIs were noted in the poorly differentiated type (P < 0.01). The survival rate was significantly lower in patients with a higher MCM7 LI (>49.5%) than in those with a lower LI (P < 0.05) at stage III-IV. However, the survival rate in the patients with a higher geminin LI (>19.5%) was significantly higher than in those with a lower LI (P < 0.05) at stage IV.

Transcription Factor POU6F1 Is Important for Proliferation of Clear Cell Adenocarcinoma of the Ovary and Is a Potential New Molecular Target

Clear cell adenocarcinoma of the ovary often shows resistance to anticancer agents. It accounts for 20% of epithelial ovarian cancer in Japan versus around 5% in other countries. We investigated new molecules to use when developing molecular-targeting therapy for clear cell adenocarcinoma. Reverse transcriptase polymerase chain reaction and Western blot analysis were performed to confirm the expression of POU6F1 in several kinds of cell lines derived from epithelial ovarian carcinoma. Microarray analyses were performed using 2 ovarian cancer microarray data sets available on the Internet. Immunohistochemical staining was also done to confirm both the expression and the localization of POU6F1 using human ovarian epithelial ovarian carcinoma tissue specimens. In addition, the gene cluster located downstream of transcription factor POU6F1 was investigated to analyze its role in the proliferation of clear cell adenocarcinoma of the ovary via the lysophosphatidic acid receptor, a G protein-coupled receptor. Furthermore, RNA interference studies with small interfering RNA (siRNA) were performed to assess the effect of POU6F1 on proliferation of xenograft tumors after injection of clear cell adenocarcinoma cells into nude mice. Expression of POU6F1 at messenger RNA and protein was confirmed in cell lines derived from epithelial ovarian carcinoma. The microarray analyses performed using the 2 ovarian cancer microarray data sets available on the Internet indicated that POU6F1 expression was significantly greater in clear cell adenocarcinoma. Immunostaining confirmed the nuclear localization of POU6F1 in clear cell adenocarcinoma (100%). Exposure to the siRNA for POU6F1 reduced the expression of lysophosphatidic acid receptors, which are G protein-coupled receptors involved in tumor cell proliferation. POU6F1 siRNA dose-dependently suppressed the proliferation of clear cell adenocarcinoma cell lines, and a similar effect was confirmed for tumors transplanted into nude mice. Clear cell adenocarcinoma shows little response to standard therapy. The results of this study suggested that the transcription factor POU6F1 could be a new molecular target for treatment of this cancer.

Expression of interleukin-1 receptor-associated kinase-1 in non-small cell lung carcinoma and preneoplastic lesions.

To identify the pattern of interleukin-1 receptor-associated kinase (IRAK-1) protein expression in non-small cell lung carcinoma (NSCLC) and corresponding preneoplastic lesions. Archived tissue from NSCLC (adenocarcinoma and squamous cell carcinoma; n = 306) and adjacent bronchial epithelial specimens (n = 315) were analyzed for the immunohistochemical expression of IRAK-1, and the findings were correlated with patients' clinicopathologic features. Furthermore, we investigated the correlation between IRAK-1 expression and expression of NF-kappaB and IL-1alpha in tumor specimens. NSCLC tumors showed significantly higher cytoplasmic and lower nuclear IRAK-1 expression than normal epithelium. Squamous dysplasias had significantly higher cytoplasmic IRAK-1 expression than normal epithelium. In tumors, a significant positive correlation was detected between IRAK-1 expression (nuclear and cytoplasmic; P = 0.011) and IL-1alpha cytoplasmic expression (P < 0.0001). The correlation between the expression of the markers and patients' clinicopathologic features varied according to tumor histologic type and sex. High IRAK-1 cytoplasmic expression correlated with worse recurrence-free survival in women with NSCLC [hazard ratio (HR), 2.204; P = 0.033], but not in men. In adenocarcinoma, combined low level of expression of nuclear IRAK-1 and NF-kappaB correlated significantly with worse overall (HR, 2.485; P = 0.007) and recurrence-free (HR, 3.058; P = 0.006) survivals in stage I/II patients. IRAK-1 is frequently expressed in NSCLC tissue specimens, and this expression is an early phenomenon in the sequential development of lung cancer. IRAK-1 is a novel inflammation-related marker and a potential target for lung cancer chemopreventive strategies.

Autocrine Bone Morphogenetic Protein-9 Signals through Activin Receptor-like Kinase-2/Smad1/Smad4 to Promote Ovarian Cancer Cell Proliferation

Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes, we characterized transforming growth factor beta/BMP receptor and Smad expression in immortalized ovarian surface epithelial cells and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signaling in ovarian cancer. Using small interfering RNA, ligand trap, inhibitor, and ligand stimulation approaches, we show that BMP9 acts as a proliferative factor for immortalized ovarian surface epithelial cells and ovarian cancer cell lines, signaling predominantly through an ALK2/Smad1/Smad4 pathway rather than through ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signaling that is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that approximately 25% of epithelial ovarian cancers express BMP9, whereas normal human ovarian surface epithelial specimens do not. Our data indicate that BMP9 signaling through ALK2 may be a novel therapeutic target in ovarian cancer.

Safety and efficacy of cytology brushings versus standard fine-needle aspiration in evaluating cystic pancreatic lesions: a controlled study

Cystic pancreatic lesions (CPLs) are increasingly detected by various imaging studies. Mucinous CPLs carry a risk of malignant transformation but this is often difficult to diagnose preoperatively. In a previous report of 10 suspected mucinous CPLs, the cellular yield of endoscopic ultrasonography (EUS)-guided cytology brushings was found to be superior to the yield from standard fine-needle aspiration (FNA). The aim of this prospective and blinded study was to compare the cytology yield of mucinous epithelium from brushing with FNA in suspected mucinous CPLs. In total, 37 patients with 39 CPLs measuring at least 20 mm were enrolled between June 2006 and July 2008 for EUS-cytobrushing and EUS-FNA of CPLs. Demographic, clinical, EUS, cytopathologic, and surgical data were recorded whenever available. Yield of cytology brushings was compared with that of FNA. Procedure morbidity was evaluated after 30 days. The main outcome assessed was yield of intracellular mucin (ICM) on cytobrushing specimens compared with EUS-FNA for the diagnosis of suspected mucinous CPL. Cytobrushings were more likely to detect ICM than the EUS-FNA method ( P = 0.001). In three patients with hypocellular FNA, dysplasia was found on cytology brushing and later confirmed by surgical pathology. Significant complications occurred in three patients (8 %): one postbrushing bleeding and two acute pancreatitis. Cytology brushings are more likely to provide an adequate mucinous epithelium specimen than standard FNA and could aid the diagnosis of CPLs in a selective group of patients.