CEACAM1 Expression Research Articles

HIF1A regulates xenophagic degradation of adherent and invasive Escherichia coli (AIEC)

The hypoxia inducible transcription factor HIF1 activates autophagy, a general catabolic pathway involved in the maintenance of cellular homeostasis. Dysfunction in both autophagy and HIF1 has been implicated in an increasing number of human diseases, including inflammatory bowel disease (IBD), such as Crohn disease (CD). Adherent invasive E. coli (AIEC) colonize ileal mucosa of CD patients and strongly promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses. Here, we aim to characterize the contribution of HIF1 in xenophagy, a specialized form of autophagy involved in the degradation of intracellular bacteria. Our results showed that endogenous HIF1A knockdown increased AIEC survival in intestinal epithelial cells. We demonstrate that the increase in survival rate correlates with a dramatic impairment of the autophagic flux at the autolysosomal maturation step. Furthermore, we show that AIEC remained within single-membrane LC3-II-positive vesicles and that they were unable to induce the phosphorylation of ULK1. These results suggested that, in the absence of HIF1A, AIEC were found within LC3-associated phagosomes. Using blocking antibodies against TLR5 and CEACAM6, the 2 well-known AIEC-bound receptors, we showed that downstream receptor signaling was necessary to mediate ULK1 phosphorylation. Finally, we provide evidence that HIF1 mediates CEACAM6 expression and that CEACAM6 is necessary to recruit ULK1 in a bacteria-containing signaling hub. Collectively, these results identify a new function for HIF1 in AIEC-dedicated xenophagy, and suggest that coactivation of autophagy and HIF1A expression may be a potential new therapy to resolve AIEC infection in CD patients.

Abstract 2003: CPE-delta-N promotes metastasis by regulating Nedd9 and Yap1 expression

Abstract Cancer mortality is often from metastatic disease rather than the direct effect of the primary tumor. Identification of metastasis inducing genes may offer valuable mechanistic insight for guiding specific therapeutic strategies. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-DELTA-N) that drives metastasis. CPE-DELTA-N mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-DELTA-N expression in these cell lines by si-RNA significantly inhibited their growth migration and invasion. To confirm these observations in vivo, an orthotopic nude mouse model was established. The mice implanted with a tumor derived from HCC cells transfected with si- CPE-DELTA-N RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-DELTA-N protein was translocated to the nucleus and upregulated the expression of neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9), through interaction with histone deacetylase (HDAC) 1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9, without effecting CPE-DELTA-N expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-DELTA-N was suppressed when Nedd9 was silenced by si-RNA. Furthermore CPE-DELTA-N protein up-regulation also increased Yes Associated Protein 1 (YAP1), Carcinoembryonic Antigen-related Cell Adhesion Molecule 5 (ceacam5) and Carbonyl Reductase 1(cbr1) and silencing CPE-DELTA-N with siRNA suppressed YAP1, CEACAM5 and CBR1 protein expression. cDNA Microarray studies of HCC cells overexpressing CPE-DELTA-N showed elevated expression of 27 genes associated with metastasis, while 30 genes associated with tumor suppressor function, which included insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. These data were further confirmed by qRTPCR.. In clinical studies, CPE-DELTA-N mRNA quantification in 99 patients with primary tumor (T) and normal surrounding tissue (N) established a T/N cut off value which predicted future intra-hepatic metastasis with high sensitivity and specificity, independent of cancer stage. Additionally, prospective study on 46 resected PHEO/PGLs having very high CPE-DELTA-N mRNA copy numbers accurately predicted future metastasis or recurrence in patients diagnosed with benign tumors at time of surgery, versus low copy numbers in post-surgery, disease-free patients. Additionally, CPE-DELTA-N is a diagnostic biomarker for metastasis in papillary thyroid carcinoma and colorectal cancer. Thus CPE-DELTA-N induces tumor metastasis and is a powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients, superior to histopathological diagnosis. Citation Format: Saravana R.K Murthy, Terence K. Lee, Niamh X. Cawley, Stephen M. Hewitt, Peng Y. Loh. CPE-delta-N promotes metastasis by regulating Nedd9 and Yap1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2014-2003

Abstract 4966: Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node

Abstract Objective: Lung cancer has become one of the leading causes of cancer-related mortality in the world. Metastasis remains the overwhelming causes of death for lung cancer patients. 80-90% of lung cancer deaths are caused by metastasis. However, an ideal in vitro model to explore the regulation and the molecules that control the lymph node metastasis of lung cancer is required. Methods: A gene expression profiling for both of the cell lines were generated by microarray. Further investigations were carried out in the lung cancer cell lines and paired tissues by migration and invasion assays and immunohistochemistry. Results: Firstly, we established a paired human lung adenocarcinoma cell lines from primary tumor site (named DC cells) and metastatic paratracheal lymph nodes (named DL cells). The two cell lines have different biological characteristics which DC cells grow clump easily and DL cells grow more scattered. The doubling time of DC and DL cell lines were 32.6h and 45.2h, respectively. The detection of the human D1S80 and D17S30 VNTR locus verified the same individual for DC and DL cell lines. Secondary, Microarray analysis revealed 232 upregulated probe sets and 739 downregulated probe sets in DL cell line compared to DC cell line. Compared to DC cells, the transcriptional expression of 27 genes were dramatically increased (p < 0.05), including AIM2, IGFBP4, IL6 and IL6R in DL cells, while expression of 55 genes was clearly decreased (p < 0.05), including FGF13, FGF19, IL1A, IL1B, MCAM, MMP15, MMP7, and TGFB2 by real-time PCR analysis. Furthermore, we selected 32 genes and screened the expression of those genes in 10-paired human NSCLC primary lung cancer tissues and matched metastasis lymph nodes tissues by real-time PCR. The results showed: 8 out of 10 metastasis lymph node tissues got up-regulated of RGS7, VNN2, and 7 out of 10 metastasis lymph node tissues got up-regulated of PECAM1, VNN1, CLIC2 expression. On the other hand, 8 of 10 metastasis lymph node tissues got down-regulated expression of CEACAM6, and 7 out of 10 got down-regulated expression of PIAS3. At mean time, none of metastasis lymph node tissues got decreased expression of TNFRSF9, which only was seen in DL cells. The further study in other lung cancer cell lines indicated that CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes influenc lung cancer cell migration ability in vitro. And these genes were also analyzed in more lung cancer tissues by immunohistochemistry staining. Conclusion: We established a paired human lung adenocarcinoma cell lines from primary tumor site and metastatic paratracheal lymph nodes. The CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes involve in the lung cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Jun Chen, Min Wang, Lingling Zu, Yongwen Li, Weiqiang Wang,Ying Li, Hongyu Liu. Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2014-4966

190P - Microrna-29A Suppresses Growth, Migration and Invasion of Lung Adenocarcinoma By Targeting Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6

ABSTRACT Aim: Carcinoembryonic antigen-related cell adhesion molecule (CEACAM)6 is the important regulator in cell adhesion, invasion and metastasis. MicroRNAs (miRNAs), a group of small non-coding RNAs that regulate the expression of many genes can act as oncogenes and tumor suppressors. The aim of this study was to evaluate the functional roles of CEACAM6 in lung adenocarcinoma and to identify miRNAs that could inhibits the growth, migration and invasion of lung adenocarcinoma by targeting CEACAM6. Methods: The expression of CEACAM6 in lung adenocarcinoma tissues was detected by immunohistochemistry. Cell proliferation, migration and invasion assays in A549 cell lines were performed in vitro and the A549 xenograft models were used for in vivo analysis. Bioinformatics prediction, western blot and luciferase assay were identified the target of miRNA. Results: Patients with low expression of CEACAM6 protein had significant longer overall survival, compared with those with high expression of CEACAM6 protein. The knockdown of CEACAM6 suppressed cell proliferation, migration and invasion, whereas CEACAM6 overexpression increased proliferation, migration and invasion in vitro. These findings are consistent with results of in vivo xenograft experiments. The relative luciferase activity was significantly decreased by miR-29a that was potentially targeting CEACAM6 using miRNA target predicting program in A549 cells co-transfected with wild type CEACAM6-3'-UTR, whereas the luciferase activity was not suppressed by the vector containing the mutant 3'-UTR or negative vector. Furthermore, miR-29a suppressed cell proliferation, migration and invasion in vitro and inhibited tumor growth of lung adenocarcinoma in vivo. Conclusions: miR-29a can suppress tumor growth, migration and invasion of lung adenocarcinoma by targeting CEACAM6, which may provide a potential therapeutic target for treatment of lung adenocarcinoma. Disclosure: All authors have declared no conflicts of interest.

CEACAM5 has different expression patterns in gastric non-neoplastic and neoplastic lesions and cytoplasmic staining is a marker for evaluation of tumor progression in gastric adenocarcinoma

ObjectiveThe aim of this study was to investigate the expression patterns of CEACAM5 in non-neoplastic and neoplastic gastric lesions, as well as its application in the differential diagnosis and its relationship with tumor progression. MethodsCEACAM5 expression was detected by immunohistochemical staining in the serial sections of the gastric neoplastic and non-neoplastic lesions. The impacts of CEACAM5 expression patterns on tumor progression were evaluated by statistics, the clinical and pathological data included sex, age, tumor extension, lymph node involvement and tumor staging. ResultsThere was no CEACAM5 expression in normal gastric epithelial cells. In hyperplastic polyps, CEACAM5 was expressed with apical membranous staining in the hyperplastic and prolonged gastric pit adjacent to the surface. Intestinal metaplasia (IM) expressed CEACAM5 mainly with membranous pattern, and some cases showed membranous staining mixed with cytoplasmic staining. GIN expressed CEACAM5 mainly with membranous staining, but the mixed staining of cytoplasmic and membranous patterns increased, and especially in the high grade GIN, cytoplasmic staining of CEACAM5 began to occur. Compared with IM and GIN, CEACAM5 expression patterns of hyperplastic polyp showed a significant difference (P=0.000). IM, low grade GIN and the whole GIN showed no significant difference in CEACAM5 expression patterns (P=0.355), but IM and high grade GIN showed a significant difference (P=0.027). There was a significant difference between low and high grade GIN (P=0.002). GIN and well-differentiated carcinomas showed no significant difference (P=0.070), but low grade GIN and well differentiated carcinomas showed a significant difference (P=0.006). In gastric adenocarcinomas, CEACAM5 expression patterns showed a significant difference in tumor grading (P=0.010) and Laurén classification (P=0.001). In histological grading, well differentiated carcinomas showed more membranous staining than moderately and poorly differentiated, and more cytoplasmic CEACAM5 staining was detected in moderately and poorly differentiated carcinomas. Similar to that, in Laurén classification, intestinal carcinomas showed more membranous staining, and diffuse carcinomas showed more cytoplasmic staining. Moreover, CEACAM5 expression patterns showed a significant difference in tumor extension (P=0.012), lymph node involvement (P=0.015) and tumor staging (P=0.002), suggesting that CEACAM5 should be involved in tumor progression. In advanced carcinomas, CEACAM5 was expressed with more cytoplasmic staining regardless of the histological classification. ConclusionCEACAM5 had different expression patterns in gastric non-neoplastic and neoplastic lesions. The CEACAM5 expression patterns were associated with tumor progression. Membranous staining of CEACAM5 might be a marker of premalignancy in gastric lesions, and cytoplasmic CEACAM5 might enhance tumor invasion and migration and be an evaluated marker for progressive and advanced gastric cancer. Also, it might be useful for the differential diagnosis of gastric premalignant lesions.

Diet-induced hypoxia responsive element demethylation increases CEACAM6 expression, favouring Crohn's disease-associated Escherichia coli colonisation

ObjectiveAdherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC strains adhere to enterocytes via interaction between type 1 pili and CEACAM6 receptors abnormally expressed on...

Molecular Profiling of Premalignant Lesions in Lung Squamous Cell Carcinomas Identifies Mechanisms Involved in Stepwise Carcinogenesis

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.

HS3ST2 modulates breast cancer cell invasiveness via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and cadherin expression.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.

A Marker of Endocrine Receptor-Positive Cells, CEACAM6, Is Shared by Two Major Classes of Breast Cancer: Luminal and HER2-Enriched

Elucidating the phenotypic evolution of breast cancer through distinct subtypes relies heavily on defining a lineage blueprint of the normal human breast. Here, we show that in normal breast, within the luminal epithelial lineage, a subset of cells characterized by strong staining for endocrine receptors are also characterized by expression of the surface marker CEACAM6. Topographically, this pattern of staining predominates in terminal ductal lobular units, rather than in interlobular ducts. In culture, CEACAM6-expressing cells remain essentially postmitotic under conditions in which the other cells of luminal epithelial lineage are highly proliferative. We examined the pattern of expression among three major breast cancer subtypes: luminal, HER2-enriched, and basal-like. In 104 biopsies, the luminal and HER2-enriched subtypes showed a high proportion of CEACAM6(+) tumors (78% and 83%, respectively); the basal-like subtype showed a low proportion (28%). Further accentuation of this pattern was observed in 13 established breast cancer cell lines. When differentiation was induced by all-trans retinoic acid, CEACAM6 expression strongly correlated with luminal-like differentiation. Furthermore, CEACAM6(+) cancer cells were less proliferative than CEACAM6(-) cells in tumorsphere assays and were less tumorigenic in nude mice. Based on these observations, we propose that luminal and HER2-enriched breast cancers are more closely related than previously thought and may share a common cell of origin.

OP019 Hypoxia responsive elements methylation status governs adherent-invasive Escherichia coli -receptor CEACAM6 expression in Crohn's disease

OP019 Hypoxia responsive elements methylation status governs adherent-invasive Escherichia coli -receptor CEACAM6 expression in Crohn's disease

Influences of enteral nutrition upon CEACAM6 expression by intestinal epithelial cells

Exclusive enteral nutrition is established as an initial therapy to induce remission in active Crohn's disease (CD), especially in children, but the mechanisms of action of this therapy are yet to be fully defined. CEACAM6 protein is an adhesion molecule that is up-regulated in active CD and implicated in the attachment of adherent-invasive Escherichia coli (AIEC) to the gut epithelium. Using the Caco-2 human adenocarcinoma cell line, this study showed that the incubation of human cells with a polymeric formula (PF) resulted in a dose-dependent increase in the expression of CEACAM6, and that this effect was most noticeable on the cell surface. Further investigation revealed that PF doubled the release of CEACAM6 protein by Caco-2 cells exposed to PF, and that an increase in release of soluble CEACAM6 inversely correlated with the ability of AIEC to associate with the intestinal epithelial cells. Our findings suggest that the secretion of cell surface-associated proteins acting as releasable decoys may be an aspect of the gut's innate immune response to pathogenic bacteria that is strengthened by PF in the setting of CD.

Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons

BackgroundThe carcinoembryonic antigen (CEA)-related cell adhesion molecules CEACAM1 (BGP, CD66a), CEACAM5 (CEA, CD66e) and CEACAM6 (NCA, CD66c) are expressed in human lung. They play a role in innate and adaptive immunity and are targets for various bacterial and viral adhesins. Two pathogens that colonize the normally sterile lower respiratory tract in patients with chronic obstructive pulmonary disease (COPD) are non-typable Haemophilus influenzae (NTHI) and Moraxella catarrhalis. Both pathogens bind to CEACAMs and elicit a variety of cellular reactions, including bacterial internalization, cell adhesion and apoptosis.MethodsTo analyze the (co-) expression of CEACAM1, CEACAM5 and CEACAM6 in different lung tissues with respect to COPD, smoking status and granulocyte infiltration, immunohistochemically stained paraffin sections of 19 donors were studied. To address short-term effects of cigarette smoke and acute inflammation, transcriptional regulation of CEACAM5, CEACAM6 and different CEACAM1 isoforms by cigarette smoke extract, interferons, Toll-like receptor agonists, and bacteria was tested in normal human bronchial epithelial (NHBE) cells by quantitative PCR. Corresponding CEACAM protein levels were determined by flow cytometry.ResultsImmunohistochemical analysis of lung sections showed the most frequent and intense staining for CEACAM1, CEACAM5 and CEACAM6 in bronchial and alveolar epithelium, but revealed no significant differences in connection with COPD, smoking status and granulocyte infiltration. In NHBE cells, mRNA expression of CEACAM1 isoforms CEACAM1-4L, CEACAM1-4S, CEACAM1-3L and CEACAM1-3S were up-regulated by interferons alpha, beta and gamma, as well as the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C). Interferon-gamma also increased CEACAM5 expression. These results were confirmed on protein level by FACS analysis. Importantly, also NTHI and M. catarrhalis increased CEACAM1 mRNA levels. This effect was independent of the ability to bind to CEACAM1. The expression of CEACAM6 was not affected by any treatment or bacterial infection.ConclusionsWhile we did not find a direct correlation between CEACAM1 expression and COPD, the COPD-associated bacteria NTHi and M. catarrhalis were able to increase the expression of their own receptor on host cells. Further, the data suggest a role for CEACAM1 and CEACAM5 in the phenomenon of increased host susceptibility to bacterial infection upon viral challenge in the human respiratory tract.

Expression of human CEACAM1 in transgenic mice limits the Opa-specific immune response against meningococcal outer membrane vesicles

Outer membrane vesicles (OMVs) have been extensively investigated as meningococcal vaccine candidates. Among their major components are the opacity (Opa) proteins, a family of surface-exposed outer membrane proteins important for bacterial adherence and entry into host cells. Many Opa-dependent interactions are mediated through the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of receptors. Importantly, binding of Opa to CEACAM1 has been reported to suppress human CD4 T cell proliferation in vitro in response to OMV preparations. This raises the question whether OMV vaccines should contain Opa proteins at all. Until now it has been difficult to answer this question, as the proposed immunosuppressive effect was only demonstrated with human cells in vitro, while immunization experiments in mice are not informative because the Opa interaction is specific for human CEACAM1. In the present study we have used Opa+ and Opa− OMVs for immunization experiments in a human CEACAM1 transgenic mouse model. OMVs were prepared from a meningococcal strain H44/76 variant expressing the CEACAM1-binding OpaJ protein, and from an isogenic variant in which all opa genes have been inactivated. Both the CEACAM1 expressing transgenic mice and their congenic littermates lacking it were immunized twice with the OMV preparations, and the sera were analyzed for bactericidal activity and ELISA antibody titres. Total IgG antibodies against the OMVs were similar in both mouse strains. Yet the titres for IgG antibodies specific for purified OpaJ protein were significantly lower in the mice expressing human CEACAM1 than in the nontransgenic mice. No significant differences were found in bactericidal titres among the four groups. Overall, these data indicate that expression of human CEACAM1 confers a reduced Opa-specific antibody response in vivo without affecting the overall immune response against other OMV antigens.

Clinical and experimental studies regarding the expression and diagnostic value of carcinoembryonic antigen-related cell adhesion molecule 1 in non-small-cell lung cancer

BackgroundCarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a multifunctional Ig-like cell adhesion molecule that has a wide range of biological functions. According to previous reports, serum CEACAM1 is dysregulated in different malignant tumours and associated with tumour progression. However, the serum CEACAM1 expression in non-small-cell lung carcinomas (NSCLC) is unclear. The different expression ratio of CEACAM1-S and CEACAM1-L isoform has seldom been investigated in NSCLC. This research is intended to study the serum CEACAM1 and the ratio of CEACAM1-S/L isoforms in NSCLC.MethodsThe expression of the serum CEACAM1 was determined by enzyme-linked immunosorbent assay. The protein expression and the location of CEACAM1 in tumours were observed by immunohistochemical staining. The CEACAM1 mRNA levels in tumour and normal adjacent tissues were measured using quantitative real-time PCR, and the expression patterns and the rate of CEACAM1-S and CEACAM1-L were analysed by reverse transcription-PCR.ResultsSerum CEACAM1 levels were significantly higher in NSCLC patients compared with that from normal healthy controls (P <0.0001). 17 patients (81%) among 21 showed high expression of CEACAM1 by immunohistochemical staining. Although no significant differences were found between tumour and normal tissues on mRNA expression levels of CEACAM1 (P >0.05), the CEACAM1-S and the CEACAM1-S/L (S: L) ratios were significantly higher in tumour than normal tissues (P <0.05).ConclusionsOur data indicated that the serum levels of CEACAM1 could discriminate lung cancer patients from health donors and that CEACAM1 might be a useful marker in early diagnosis of NSCLC. Moreover, our results showed that the expression patterns of CEACAM1 isoforms could be changed during oncogenesis, even when total CEACAM1 in tumour tissues did not show significant changes. Our study suggested that the expression ratios of CEACAM1-S/CEACAM1-L might be a better diagnostic indicator in NSCLC than the quantitative changes of CEACAM1.

In Vivo Adaptation and Persistence of Neisseria meningitidis within the Nasopharyngeal Mucosa

Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa.

Regulation of the Epithelial Adhesion Molecule CEACAM1 Is Important for Palate Formation

Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1 −/−) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1 −/− mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1−/−mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.

Prostate derived Ets transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 constitute a highly active oncogenic axis in breast cancer

We previously reported overexpression of Prostate derived Ets transcription factor (PDEF) in breast cancer and its role in breast cancer progression, supporting PDEF as an attractive target in this cancer. The goal of this research was to identify specific PDEF induced molecules that, like PDEF, show overexpression in breast tumors and a role in breast tumor progression. PDEF expression was down regulated by shRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulated and control MCF-7 cells were used to screen the HG-U133A human gene chips. These analyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67% of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF and CEACAM6 expression was elevated 10-fold or higher in comparison to normal breast tissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold or higher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failed to show concordant elevated expression with PDEF in primary tumors. To determine the significance of elevated PDEF and CEACAM6 expression to tumor phenotype, their expression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenic role for both PDEF and CEACAM6 in breast cancer. Together, these findings show that PDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest that targeting of these molecules should provide novel treatments for most breast cancer patients.

Clinical applications of gene expression in colorectal cancer.

Despite developments in diagnosis and treatment, 20% of colorectal cancer (CRC) patients present with metastatic disease and 30% of cases recur after curative surgery. Furthermore, the molecular factors involved in prognosis and response to therapy in CRC is poorly understood. The aims of this study were to quantitatively examine the expression of target genes in colorectal cancer and to correlate their expression levels with clinico-pathological variables. A detailed analysis of published CRC microarray data was performed to identify the most prominent genes. The selected genes were validated in fifty-two pairs of fresh colorectal tumour and associated normal tissue specimens by RQ-PCR using TaqMan(®) assays. Statistical analysis and correlation with clinicopathological data was performed using SPSS software. Expression levels of CXCL12 (P=0.000), CDH17 (P=0.026), MUC2 (P=0.000), L-FABP (P=0.000) and PDCD4 (P=0.000) were down regulated and IL8 (P=0.000) was upregulated in tumours compared to normal colorectal tissues. No significant differences were noted in expression of CEACAM5, CXCR4, CXCR7, TGFB1, TGFBR1 and TGFBR2. Furthermore, we found significant associations of gene expression levels and clinicopathological variables such as tumour size, grade, invasion and lymph node status. We identified a comprehensive list of genes with highly differential expression patterns in colorectal cancer that could serve as molecular markers to complement existing histopathological factors in diagnosis, follow up and therapeutic strategies for individualised care of patients.

Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts

We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε−/−) mice to examine this mechanistically. PKCε−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.

Intestinal Persistence of Crohnʼs Disease-associated Adherent-Invasive Escherichia Coli Enhances Spontaneous Visceral Hypersensitivity

Crohn's disease (CD) results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both genetic and environmental influencing factors. Various studies in CD patients have reported abnormal gut colonization by pathogenic Escherichia coli referred to as adherent invasive E. coli (AIEC) linked to overexpression of the bacterial colonizing receptor CEACAM6. Among characteristics of CD are the presence of colonic hypersensitivity and abdominal pain. The aim of our study was to investigate whether, intestinal AIEC colonization could be at the origin of spontaneous development of visceral hypersensitivity and whether this latter may persist even after bacterial clearance and intestinal inflammation alleviation. Transgenic mice overexpressing the human CEACAM6 glycoprotein and their wild-type littermates were orally infected by CD-associated AIEC bacteria (reference strain LF82) or by a non-pathogenic E. coli (MG1655). At 3, 9 and 21 days after infection, visceral hypersensitivity was assessed using a technique based on measuring electromyographic abdominal contractions induced by colorectal distension. Briefly, by progressive inflation of a balloon inserted into the colon, stomach cramps whose amplitude is proportional to the colon sensitivity were induced. Inflammation was monitored using anatomical indicators of colitis such as spleen and colon weight, and assessed by determination of the colonic myeloperoxidase (MPO) activity and expression of systemic pro-inflammatory cytokines. Human CEACAM6 expression in transgenic mice induced a significant decrease in the visceral hypersensitivity in comparison to their housekeeping WT mice. Otherwise, infection with CD-associated AIEC LF82, but not with non-pathogenic E. coli MG1655, highly increased levels of visceral hypersensitivity at day 3 post-infection in comparison to their housekeeping WT. Of interest, such visceral hypersensitivity persisted in transgenic mice after bacterial clearance and did not match with intestinal inflammation. In the present study, we show a direct involvement of CD-associated AIEC LF82 colonization in colonic hypersensitivity. This was observed only in transgenic mice expressing CEACAM6, indicating that increased AIEC intestinal persistence may impact on colonic sensitivity, which persists after bacterial clearance. Thus hypersensitivity and abdominal pain in CD patients could be linked to bacterial infection.