EMP2 Is a Novel Regulator of Stemness in Breast Cancer Cells.

Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24-, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.

Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not.

MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4.

Monocarboxylate transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here, we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export that when inhibited, enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors, further supporting their use as anti-cancer therapeutics.

Microgeographic Proteomic Networks of the Human Colonic Mucosa and Their Association With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Interactions between mucosal cell types, environmental stressors, and intestinal microbiota contribute to pathogenesis in inflammatory bowel disease (IBD). Here, we applied metaproteomics of the mucosal-luminal interface to study the disease-related biology of the human colonic mucosa. METHODS: We recruited a discovery cohort of 51 IBD and non-IBD subjects endoscopically sampled by mucosal lavage at 6 colonic regions, and a validation cohort of 38 no-IBD subjects. Metaproteome data sets were produced for each sample and analyzed for association with colonic site and disease state using a suite of bioinformatic approaches. Localization of select proteins was determined by immunoblot analysis and immunohistochemistry of human endoscopic biopsy samples. RESULTS: Co-occurrence analysis of the discovery cohort metaproteome showed that proteins at the mucosal surface clustered into modules with evidence of differential functional specialization (eg, iron regulation, microbial defense) and cellular origin (eg, epithelial or hemopoietic). These modules, validated in an independent cohort, were differentially associated spatially along the gastrointestinal tract, and 7 modules were associated selectively with non-IBD, ulcerative colitis, and/or Crohn's disease states. In addition, the detailed composition of certain modules was altered in disease vs healthy states. We confirmed the predicted spatial and disease-associated localization of 28 proteins representing 4 different disease-related modules by immunoblot and immunohistochemistry visualization, with evidence for their distribution as millimeter-scale microgeographic mosaic. CONCLUSIONS: These findings suggest that the mucosal surface is a microgeographic mosaic of functional networks reflecting the local mucosal ecology, whose compositional differences in disease and healthy samples may provide a unique readout of physiologic and pathologic mucosal states.

High expression of AGR2 in lung cancer is predictive of poor survival.

BACKGROUND: Anterior gradient 2 (AGR2) is a protein disulfide isomerase-like protein widely expressed in many normal tissues as well as cancers. In our study, non-neoplastic bronchial epithelial cells as well as non-small cell lung cancer (NSCLC) cells express AGR2 protein. METHODS: AGR2 expression was analyzed on lung tissue microarrays. Tumor staining was correlated with clinical outcomes. RESULTS: On a lung cancer tissue microarray using immunohistochemistry, expression levels in cancer showed generally decreasing intensities in order from adenocarcinomas with mucinous components, other adenocarcinomas, squamous carcinomas, to large cell carcinomas. The study cohort was comprised of 400 cases. As a group, there was a slight trend of lower expression with increasing tumor grade. AGR2 expression level was a significant predictor of overall survival in younger patients only. Patients under 65 with lower levels showed a significantly better survival for both men and women. Patients over 65, in contrast, showed no such trend. CONCLUSIONS: Nearly all NSCLC tumors show AGR2 expression. Lung cancer expression of AGR2 has prognostic value for younger patients.

Ribonucleotide reductase subunit M2 predicts survival in subgroups of patients with non-small cell lung carcinoma: effects of gender and smoking status.

BACKGROUND: Ribonucleotide reductase catalyzes the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. The functional enzyme consists of two subunits - one large (RRM1) and one small (RRM2 or RRM2b) subunit. Expression levels of each subunit have been implicated in prognostic outcomes in several different types of cancers. EXPERIMENTAL DESIGN: Immunohistochemistry for RRM1 and RRM2 was performed on a lung cancer tissue microarray (TMA) and analyzed. 326 patients from the microarray were included in this study. RESULTS: In non-small cell lung cancer (NSCLC), RRM2 expression was strongly predictive of disease-specific survival in women, non-smokers and former smokers who had quit at least 10 years prior to being diagnosed with lung cancer. Higher expression was associated with worse survival. This was not the case for men, current smokers and those who had stopped smoking for shorter periods of time. RRM1 was not predictive of survival outcomes in any subset of the patient group. CONCLUSION: RRM2, but not RRM1, is a useful predictor of survival outcome in certain subsets of NSCLC patients.

Biologic roles of estrogen receptor-ß and insulin-like growth factor-2 in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) occurs in 10-15% of patients yet accounts for almost half of all breast cancer deaths. TNBCs lack expression of estrogen and progesterone receptors and HER-2 overexpression and cannot be treated with current targeted therapies. TNBCs often occur in African American and younger women. Although initially responsive to some chemotherapies, TNBCs tend to relapse and metastasize. Thus, it is critical to find new therapeutic targets. A second ER gene product, termed ERß, in the absence of ERα may be such a target. Using human TNBC specimens with known clinical outcomes to assess ERß expression, we find that ERß1 associates with significantly worse 5-year overall survival. Further, a panel of TNBC cell lines exhibit significant levels of ERß protein. To assess ERß effects on proliferation, ERß expression in TNBC cells was silenced using shRNA, resulting in a significant reduction in TNBC proliferation. ERß-specific antagonists similarly suppressed TNBC growth. Growth-stimulating effects of ERß may be due in part to downstream actions that promote VEGF, amphiregulin, and Wnt-10b secretion, other factors associated with tumor promotion. In vivo, insulin-like growth factor-2 (IGF-2), along with ERß1, is significantly expressed in TNBC and stimulates high ERß mRNA in TNBC cells. This work may help elucidate the interplay of metabolic and growth factors in TNBC.

SETDB1 accelerates tumourigenesis by regulating the WNT signalling pathway.

We investigated the oncogenic role of SETDB1, focusing on non-small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT-ß-catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1.

Epithelial membrane protein-2 (EMP2) activates Src protein and is a novel therapeutic target for glioblastoma.

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvß3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.

Rationale and preclinical efficacy of a novel anti-EMP2 antibody for the treatment of invasive breast cancer.

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer worldwide is still unacceptably high. Thus, there is an urgent need to discover new molecular targets. In this article, we show evidence for a novel target in human breast cancer, the tetraspan protein epithelial membrane protein-2 (EMP2). Using tissue tumor arrays, protein expression of EMP2 was measured and found to be minimal in normal mammary tissue, but it was upregulated in 63% of invasive breast cancer tumors and in 73% of triple-negative tumors tested. To test the hypothesis that EMP2 may be a suitable target for therapy, we constructed a fully human immunoglobulin G1 (IgG1) antibody specific for a conserved domain of human and murine EMP2. Treatment of breast cancer cells with the anti-EMP2 IgG1 significantly inhibited EMP2-mediated signaling, blocked FAK/Src signaling, inhibited invasion, and promoted apoptosis in vitro. In both human xenograft and syngeneic metastatic tumor monotherapy models, anti-EMP2 IgG1 retarded tumor growth without detectable systemic toxicity. This antitumor effect was, in part, attributable to a potent antibody-dependent cell-mediated cytotoxicity response as well as direct cytotoxicity induced by the monoclonal antibody. Together, these results identify EMP2 as a novel therapeutic target for invasive breast cancer.

Prostate cancer cell phenotypes based on AGR2 and CD10 expression.

The combination of expression patterns of AGR2 (anterior gradient 2) and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10(low)AGR2(high), CD10(high)AGR2(high), CD10(low)AGR2(low), and CD10(high)AGR2(low) were distinguished. AGR2(+) tumors were associated with longer recurrence-free survival and CD10(+) tumors with shorter recurrence-free survival. In high-stage cases, the CD10(low)AGR2(high) phenotype was associated with a ninefold higher recurrence-free survival than the CD10(high)AGR2(low) phenotype. The CD10(high)AGR2(high) and CD10(low)AGR2(low) phenotypes were intermediate. The CD10(high)AGR2(low) phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10(high)AGR2(low) phenotype in primary tumors is predictive of poor outcome; however, the CD10(low)AGR2(high) phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.

Fibulin-3 as a blood and effusion biomarker for pleural mesothelioma.

BACKGROUND: New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker. METHODS: We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay. RESULTS: Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). Fibulin-3 preferentially stained tumor cells in 26 of 26 samples. In an overall comparison of patients with and those without mesothelioma, the receiver-operating-characteristic curve for plasma fibulin-3 levels had a sensitivity of 96.7% and a specificity of 95.5% at a cutoff value of 52.8 ng of fibulin-3 per milliliter. In a comparison of patients with early-stage mesothelioma with asbestos-exposed persons, the sensitivity was 100% and the specificity was 94.1% at a cutoff value of 46.0 ng of fibulin-3 per milliliter. Blinded validation revealed an area under the curve of 0.87 for plasma specimens from 96 asbestos-exposed persons as compared with 48 patients with mesothelioma. CONCLUSIONS: Plasma fibulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with effusion fibulin-3 levels, plasma fibulin-3 levels can further differentiate mesothelioma effusions from other malignant and benign effusions. (Funded by the Early Detection Research Network, National Institutes of Health, and others.).

Expression of thyroid transcription factor-1 in normal endometrium is associated with risk of endometrial cancer development.

Thyroid transcription factor-1 (TTF-1) is a DNA-binding protein that is mainly expressed in thyroid and lung tissue, but has also been found in gynecologic tissue. Recent studies have suggested that TTF-1 has tumor suppressor function in lung adenocarcinoma models. In the current study, we examined whether expression of TTF-1 in benign endometrium and endometrial hyperplasia might impact on the risk of developing endometrial cancer. Formalin-fixed paraffin-embedded endometrial tissues obtained from 535 cases were used to construct an endometrial tissue microarray. One hundred fifty of 207 patients had multiple serial endometrial specimens including 46 patients who progressed to endometrial cancer. The tissue microarray included a range of histopathologies including benign endometrium (n=231), simple hyperplasia (n=105), complex hyperplasia (n=36), simple atypical hyperplasia (n=10), complex atypical hyperplasia (n=44), and endometrial carcinoma (n=109). Expression of TTF-1 by immunohistochemistry in benign endometrium and endometrial hyperplasia was correlated with progression to cancer and clinical features known to be associated with increased risk of developing endometrial cancer. Carcinoma specimens showed a significantly greater expression of TTF-1 compared with benign endometrium and non-atypical hyperplasia (P=0.0007 and P=0.05). Presence of TTF-1 expression in benign endometrium was associated with a significantly decreased risk of cancer development (P=0.003, hazards ratio=0.104, 95% CI: 0.024-0.455). TTF-1 expression in hyperplasia did not significantly correlate with progression to cancer. The data from our study show that TTF-1 expression in normal endometrium is associated with a reduced risk of endometrial cancer development. This observation suggests that TTF-1 might function as a tumor suppressor in endometrial tissue. TTF-1 expression in normal endometrium could potentially provide clinically useful information as a biomarker for the risk of endometrial cancer.

Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer.

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.

Host-microbe relationships in inflammatory bowel disease detected by bacterial and metaproteomic analysis of the mucosal-luminal interface.

BACKGROUND: Host-microbe interactions at the intestinal mucosal-luminal interface (MLI) are critical factors in the biology of inflammatory bowel disease (IBD). METHODS: To address this issue, we performed a series of investigations integrating analysis of the bacteria and metaproteome at the MLI of Crohn's disease, ulcerative colitis, and healthy human subjects. After quantifying these variables in mucosal specimens from a first sample set, we searched for bacteria exhibiting strong correlations with host proteins. This assessment identified a small subset of bacterial phylotypes possessing this host interaction property. Using a second and independent sample set, we tested the association of disease state with levels of these 14 "host interaction" bacterial phylotypes. RESULTS: A high frequency of these bacteria (35%) significantly differentiated human subjects by disease type. Analysis of the MLI metaproteomes also yielded disease classification with exceptional confidence levels. Examination of the relationships between the bacteria and proteins, using regularized canonical correlation analysis (RCCA), sorted most subjects by disease type, supporting the concept that host-microbe interactions are involved in the biology underlying IBD. Moreover, this correlation analysis identified bacteria and proteins that were undetected by standard means-based methods such as analysis of variance, and identified associations of specific bacterial phylotypes with particular protein features of the innate immune response, some of which have been documented in model systems. CONCLUSIONS: These findings suggest that computational mining of mucosa-associated bacteria for host interaction provides an unsupervised strategy to uncover networks of bacterial taxa and host processes relevant to normal and disease states. (Inflamm Bowel Dis 2012;).

The role of estrogen, progesterone and aromatase in human non-small-cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths in both men and women worldwide. Despite advances in treatment, patients have few effective therapeutic options and survival rates remain low. Emerging evidence suggests that the hormones estrogen and progesterone play a key role in the progression of non-small-cell lung cancer (NSCLC). The aromatase enzyme, which is responsible for a key step in estrogen biosynthesis, elicits higher levels of estrogen in lung tumors as well as in metastases compared with nonmalignant tissues. Thus, aromatase may prove to be a key predictive biomarker for treatment of NSCLC. Epidemiologic and preclinical data show estrogens play a critical role in lung tumor development and progression. Two estrogen receptors, α and ß, are expressed in normal and in cancerous lung epithelium, and estrogen promotes gene transcription that stimulates cell proliferation and inhibits cell death. Furthermore, expression of both forms of estrogen receptor, progesterone receptor and aromatase in NSCLC specimens has been correlated with worse clinical outcomes. Combination therapies that include estrogen receptor downregulators and aromatase inhibitors are currently being assessed in Phase I-II clinical trials among patients with advanced NSCLC. Results will help guide future lung cancer management decisions, with a goal of achieving more effective and less toxic treatments for patients.

A metaproteomic approach to study human-microbial ecosystems at the mucosal luminal interface.

Aberrant interactions between the host and the intestinal bacteria are thought to contribute to the pathogenesis of many digestive diseases. However, studying the complex ecosystem at the human mucosal-luminal interface (MLI) is challenging and requires an integrative systems biology approach. Therefore, we developed a novel method integrating lavage sampling of the human mucosal surface, high-throughput proteomics, and a unique suite of bioinformatic and statistical analyses. Shotgun proteomic analysis of secreted proteins recovered from the MLI confirmed the presence of both human and bacterial components. To profile the MLI metaproteome, we collected 205 mucosal lavage samples from 38 healthy subjects, and subjected them to high-throughput proteomics. The spectral data were subjected to a rigorous data processing pipeline to optimize suitability for quantitation and analysis, and then were evaluated using a set of biostatistical tools. Compared to the mucosal transcriptome, the MLI metaproteome was enriched for extracellular proteins involved in response to stimulus and immune system processes. Analysis of the metaproteome revealed significant individual-related as well as anatomic region-related (biogeographic) features. Quantitative shotgun proteomics established the identity and confirmed the biogeographic association of 49 proteins (including 3 functional protein networks) demarcating the proximal and distal colon. This robust and integrated proteomic approach is thus effective for identifying functional features of the human mucosal ecosystem, and a fresh understanding of the basic biology and disease processes at the MLI.

Lung cancer chemoprevention with celecoxib in former smokers.

Ample studies suggest that the cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway plays a pivotal role in carcinogenesis and that COX-2 inhibition may help prevent lung cancer. Therefore, we conducted a randomized, double-blind, placebo-controlled trial of the COX-2-selective inhibitor celecoxib (400 mg bid for 6 months) in former-smokers (age ≥ 45, ≥ 30 pack-years of smoking, ≥ 1 year of sustained abstinence from smoking). We assessed the impact of celecoxib on cellular and molecular events associated with lung cancer pathogenesis; the primary endpoint was bronchial Ki-67 labeling index (Ki-67 LI) after 6 months of treatment. Of 137 randomized subjects, 101 completed both baseline and 6-month bronchoscopies and were evaluable for the primary endpoint analysis. The beneficial effect on Ki-67 LI was greater in the celecoxib arm (versus placebo) in a mixed-effects analysis (P = 0.0006), and celecoxib significantly decreased Ki-67 LI by an average of 34%, whereas placebo increased Ki-67 LI by an average of 3.8% (P = 0.04; t test). In participants who crossed over to the other study arm at 6 months (all of whom had received 6 months of celecoxib at the end of a 12 months treatment period), the decreases in Ki-67 LI correlated with a reduction and/or resolution of lung nodules on computed tomography. Celecoxib significantly reduced plasma c-reactive protein and interleukin-6 mRNA and protein and increased 15(S)-hydroxy-eicosatetraenoic acid levels in bronchoalveolar lavage (BAL) samples. The baseline ratio of COX-2 to 15-hydroxyprostaglandin dehydrogenase mRNA in BAL cells was a significant predictive marker of Ki-67 response to celecoxib (P = 0.002). Our collective findings support the continued investigation of celecoxib for lung cancer chemoprevention in former smokers at a low risk of cardiovascular disease.

Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src.

Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

Protein expression based multimarker analysis of breast cancer samples.

BACKGROUND: Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups. METHODS: We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-ß1, and TGF ß receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis. RESULTS: We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach. CONCLUSIONS: We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes.