Human carcinoembryonic antigen (CEA) is the prototypic member of a family of highly related cell surface glycoproteins that includes carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and others. CEACAM6 (formerly NCA), which belongs to the immunoglobulin superfamily, is a cell adhesion protein of the CEA family. It is normally expressed on the epithelial surfaces and on the surface of myeloid cells (CD66c). CEACAM6 is a multi-functional glycoprotein that mediates homotypic binding with other CEA family members and heterotypic binding with integrin receptors. It functions by organizing tissue architecture and regulating different signal transduction, while aberrant expression leads to the development of human malignancies. It was first discovered in proliferating cells of adenomas and hyperplastic polyps in comparison to benign colonic tissue when overexpressed on the surface of various cell types in model systems. CEACAM6 functions as a pan-inhibitor of cell differentiation and cell polarization, and it also causes distortion of tissue architecture. Moreover, overexpression of CEACAM6 modulates cancer progression through aberrant cell differentiation, anti-apoptosis, cell growth and resistance to therapeutic agents. In addition, CEACAM6 overexpression in multiple malignancies promotes cell invasion and metastasis, thereby representing an acquired advantage of tumor cells directly responsible for an invasive phenotype. This review focuses on the findings supporting the mechanisms of actions linking the oncogenic potential of CEACAM6 to the onset of cancer progression and pathogenesis, especially in breast cancer, and to validating CEACAM6 as a target to pave the way towards the design of efficient therapeutic strategies against breast cancer.

Although chronic inflammation and immune disorders are of great importance to the pathogenesis of both dementia and cancer, the pathophysiological mechanisms are not clearly understood. In recent years, growing epidemiological evidence and meta-analysis data suggest an inverse association between Alzheimer's disease (AD), which is the most common form of dementia, and cancer. It has been revealed that some common genes and biological processes play opposite roles in AD and cancer; however, the biological immune mechanism for the inverse association is not clearly defined. An unsupervised matrix decomposition two-stage bioinformatics procedure was adopted to investigate the opposite behaviors of the immune response in AD and breast cancer (BC) and to discover the underlying transcriptional regulatory mechanisms. Fast independent component analysis (FastICA) was applied to extract significant genes from AD and BC microarray gene expression data. Based on the extracted data, the shared transcription factors (TFs) from AD and BC were captured. Second, the network component analysis (NCA) algorithm in this study was presented to quantitatively deduce the TF activities and regulatory influences because quantitative dynamic regulatory information for TFs is not available via microarray techniques. Based on the NCA results and reconstructed transcriptional regulatory networks, inverse regulatory processes and some known innate immune responses were described in detail. Many of the shared TFs and their regulatory processes were found to be closely related to the adaptive immune response from dramatically different directions and to play crucial roles in both AD and BC pathogenesis. From the above findings, the opposing cellular behaviors demonstrate an invaluable opportunity to gain insights into the pathogenesis of these two types of diseases and to aid in developing new treatments.

OBJECTIVES: Autoimmune pancreatitis (AIP) is a representative IgG4-related and inflammatory disease of unknown etiology. To clarify mechanisms of carcinogenesis resulting from AIP, we focused on methylation abnormalities and KRAS mutations in AIP.
METHODS: Six tumor suppressor genes (NPTX2, Cyclin D2, FOXE1, TFPI2, ppENK, and p16) that exhibited hypermethylation in pancreatic carcinoma were selected for quantitative SYBR green methylation-specific polymerase chain reaction in 10 AIP specimens, 10 pancreatic adenocarcinoma cases without history of AIP containing carcinoma areas (CAs) and noncarcinoma areas (NCAs), and 11 normal pancreas (NP) samples. KRAS mutation in codons 12, 13, and 61 were also investigated using direct sequencing.
RESULTS: Hypermethylation events (≥10%) were identified in NPTX2, Cyclin D2, FOXE1, TFPI2, ppENK, and p16 in 1, 2, 2, 0, 2, and 0 CA cases, respectively, but not in these 6 candidate genes in AIP, NCA, and NP. However, the TFPI2 methylation ratio was significantly higher in AIP than NCA and NP. Direct sequencing results for KRAS showed no single-point mutations in AIP.
CONCLUSIONS: These are the first studies characterizing methylation abnormalities in AIP. AIP's inflammatory condition may be related to carcinogenesis. Further study will elucidate methylation abnormalities associated with carcinogenesis in AIP.

Construction of efficient doxorubicin (DOX) delivery systems addressing a cascade of physiological barriers remains a great challenge for better therapeutic efficacy of tumors. Herein, we design well-defined enzyme-responsive peptide-linked block copolymer, PEG-GPLGVRGDG-P(BLA-co-Asp) [PEG and P(BLA-co-Asp) are poly(ethylene glycol) and partially hydrolyzed poly(β-benzyl l-aspartate) (PBLA), respectively] (P3), with modular functionality for efficient delivery of DOX. The block copolymers were successfully obtained via click reaction to introduce peptide (alkynyl-GPLGVRGDG) into the end of PEG for initiating ring-opening polymerization of β-benzyl l-aspartate N-carboxyanhydride (BLA-NCA) by terminal amino groups followed by partial hydrolysis of PBLA segments. P3 micelle was demonstrated to encapsulate DOX efficiently through synergistic effect of benzyl group-based hydrophobic and carboxyl moiety-based electrostatic interactions. Effective matrix metalloproteinase-2 (MMP-2)-triggered cleavage of peptide for dePEGylation of P3 micelles was confirmed and residual RGD ligands were retained on the surfaces. Against HT1080 cells overexpressing MMP-2, DOX-loaded P3 micelles showed approximately 4-fold increase of the cellular internalization amount as compared with free DOX and half maximal inhibitory concentration (IC

PURPOSE: The tumor genomic copy number profile is of prognostic significance in neuroblastoma patients. We have studied the genomic copy number profile of cell-free DNA (cfDNA) and compared this with primary tumor arrayCGH (aCGH) at diagnosis.
EXPERIMENTAL DESIGN: In 70 patients, cfDNA genomic copy number profiling was performed using the OncoScan platform. The profiles were classified according to the overall pattern, including numerical chromosome alterations (NCA), segmental chromosome alterations (SCA), and MYCN amplification (MNA).
RESULTS: Interpretable and dynamic cfDNA profiles were obtained in 66 of 70 and 52 of 70 cases, respectively. An overall identical genomic profile between tumor aCGH and cfDNA was observed in 47 cases (3 NCAs, 22 SCAs, 22 MNAs). In one case, cfDNA showed an additional SCA not detected by tumor aCGH. In 4 of 8 cases with a silent tumor aCGH profile, cfDNA analysis revealed a dynamic profile (3 SCAs, 1 NCA). In 14 cases, cfDNA analysis did not reveal any copy number changes. A total of 378 breakpoints common to the primary tumor and cfDNA of any given patient were identified, 27 breakpoints were seen by tumor aCGH, and 54 breakpoints were seen in cfDNA only, including two cases with interstitial IGFR1 gains and two alterations targeting TERT CONCLUSIONS: These results demonstrate the feasibility of cfDNA copy number profiling in neuroblastoma patients, with a concordance of the overall genomic profile in aCGH and cfDNA dynamic cases of 97% and a sensitivity of 77%, respectively. Furthermore, neuroblastoma heterogeneity is highlighted, suggesting that cfDNA might reflect genetic alterations of more aggressive cell clones. Clin Cancer Res; 22(22); 5564-73. ©2016 AACRSee related commentary by Janku and Kurzrock, p. 5400.

PURPOSE: Trastuzumab emtansine (T-DM1) is indicated for previously treated HER2-positive metastatic breast cancer. Ethnic sensitivity assessment of T-DM1 was conducted using data from eight clinical studies to ensure that the clinically recommended dose is appropriate across ethnicities.
METHODS: Four approaches were used: (1) non-compartmental analysis (NCA) comparing pharmacokinetic parameters of T-DM1 and relevant analytes across ethnic groups, (2) population pharmacokinetic (popPK) analysis assessing the impact of ethnicity on pharmacokinetics, (3) comparison of T-DM1 pharmacokinetics in Japanese patients versus the global population, and (4) exposure-response analyses assessing the impact of ethnicity on safety and efficacy.
RESULTS: NCA pharmacokinetic parameters (T-DM1, total trastuzumab, DM1) were comparable across ethnic groups; mean cycle 1 T-DM1 AUCinf was 475, 442, and 518 day µg/mL for white (n = 461), Asian (n = 68), and others (n = 57), respectively. PopPK analysis showed that ethnicity (white, Asian, and others) was not a significant covariate for T-DM1 pharmacokinetics (n = 671). Additionally, visual predictive check plots indicated that observed pharmacokinetic profiles in Japanese patients (n = 42) were within the prediction interval generated from the final PopPK model. Exposure-response analyses showed that ethnicity was not a significant covariate impacting efficacy or hepatotoxicity risk, but there was a trend of greater thrombocytopenia risk among Asians versus non-Asians, which could not be explained by similar exposure between the ethnic groups. Most Asians with thrombocytopenia were able to continue T-DM1 using dose-adjustment rules recommended for the global population.
CONCLUSIONS: These results suggest that T-DM1 pharmacokinetics are comparable across ethnic groups and that use of the current dosing regimen is appropriate across ethnicities.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that regulate genes at the post-transcriptional level in spatiotemporal manner. Several miRNAs are identified as prognostic and diagnostic markers in many human cancers. Estimation of the temporal activities of the miRNAs is an important step in the way to understand the complex interactions of these important regulatory elements with transcription factors (TFs) and target genes (TGs). However, current research on miRNA activities excludes network dynamics from the studies, disregarding the important element of time in the regulatory network analysis.
RESULTS: In the current study, we combined experimentally verified miRNA-TG interactions with breast cancer microarray TG expression data to identify key miRNAs and compute their temporal activity using network component analysis (NCA). The computed activities showed that miRNAs were regulated in a time dependent manner. Our results allowed constructing a synergistic network of miRNAs using the computed miRNA activities and their shared regulation of TGs. We further extended this network by incorporating miRNA-TG, miRNA-TF, TF-miRNA and TF-TG regulations in the context of breast cancer. Our integrated network identified several miRNAs known to be involved in breast cancer regulation and revealed several novel miRNAs. Our further analysis detected substantial involvement of the miRNAs miR-324, miR-93, miR-615 and miR-1 in breast cancer, which was not known previously. Next, combining our integrated networks with functional annotation of differentially expressed genes resulted in new sub-networks. These sub-networks allowed us to identify the key miRNAs and their interactions with TFs and TGs of several biological processes involved in breast cancer. The identified markers are validated for their potential as prognostic markers for breast cancer through survival analysis.
CONCLUSIONS: Our dynamical analysis of the miRNA interactions greatly helps to discover new network based markers, and is highly applicable (but not limited) to cancer research.

Carcinoembryonic antigen (CEA), an oncofetal cell surface glycoprotein, has been widely used as a human tumor marker due to its high expression in tumors and secretion to serum. It belongs to the immunoglobulin superfamily named CEA-related cell adhesion molecule (CEACAM) family. Members of this family are detected in various cancers and have been shown to be involved in cancer growth and invasion. In this study, we examined the mRNA expression profiles of CEACAM family members including CEACAM1, CEACAM3, CEACAM4, CEACAM5 (CEA), CEACAM6, CEACAM7, and CEACAM8 in various tumor cell lines. Our screening data indicated that the mRNA expression patterns of CEACAMs in TT cells, which are derived from medullary thyroid carcinoma (MTC), were distinct from other tumor cell lines. Additionally, CEACAM4 was only expressed in TT cells, in which two novel splice variants of CEACAM4 were expressed. These findings suggested that production of CEA and CEA-related molecules in MTC may be distinct from other tumor-based production of those molecules and that the specific expression of CEACAM4 would make possible to differentiate between MTC and other CEA-producing tumors.

Pituitary adenoma results from accumulation of multiple genetic and/or epigenetic aberrations such as GNAS, MEN1, CNC, and FIPA. LRRC4 is relatively tissue-specific expressed gene in the normal brain and downregulated expression in glioma (87.5%), meningioma (80.9%), and pituitary adenoma (85.5%). It has been suggested that the aberrant expression of LRRC4 contributes to tumorigenesis in glioma. However, little is known yet about association between LRRC4 and risk of pituitary adenoma. In this study, we genotyped three LRRC4 haplotype-tagging SNPs (htSNP) by direct sequencing in case-control studies, which included 183 Han Chinese patients diagnosed with pituitary adenoma and 183 age-, gender-matched, and geographically matched Han Chinese controls. Haplotypes were reconstructed according to the genotyping data and linkage disequilibrium status of the htSNP. We observed statistically significant differences regarding the genotype TT + CT of rs6944446 in the NCA. Haplotype AC of rs3823994-rs6944446 is suggested to have a protective effect in the development of pituitary adenoma (OR 0.339; 95% CI 0.123-0.934). However, haplotype GT of rs3808058-rs6944446 (OR 1.575; 95% CI 1.048-2.368) and AGT of rs3823994-rs6944446-rs3808058 (OR 1.673; 95% CI 1.056-2.651) might be a risk factor for pituitary adenoma development. In a brief, the results support the hypothesis that polymorphisms or haplotypes in the LRRC4 may have important research significance and could be used to predict the risk of pituitary adenoma.

Understanding gene transcription regulatory networks is critical to deciphering the molecular mechanisms of different cellular states. Most studies focus on static transcriptional networks. In the current study, we used the gastrin-regulated system as a model to understand the dynamics of transcriptional networks composed of transcription factors (TFs) and target genes (TGs). The hormone gastrin activates and stimulates signaling pathways leading to various cellular states through transcriptional programs. Dysregulation of gastrin can result in cancerous tumors, for example. However, the regulatory networks involving gastrin are highly complex, and the roles of most of the components of these networks are unknown. We used time series microarray data of AR42J adenocarcinoma cells treated with gastrin combined with static TF-TG relationships integrated from different sources, and we reconstructed the dynamic activities of TFs using network component analysis (NCA). Based on the peak expression of TGs and activity of TFs, we created active sub-networks at four time ranges after gastrin treatment, namely immediate-early (IE), mid-early (ME), mid-late (ML) and very late (VL). Network analysis revealed that the active sub-networks were topologically different at the early and late time ranges. Gene ontology analysis unveiled that each active sub-network was highly enriched in a particular biological process. Interestingly, network motif patterns were also distinct between the sub-networks. This analysis can be applied to other time series microarray datasets, focusing on smaller sub-networks that are activated in a cascade, allowing better overview of the mechanisms involved at each time range.

Reliable inference of transcription regulatory networks is a challenging task in computational biology. Network component analysis (NCA) has become a powerful scheme to uncover regulatory networks behind complex biological processes. However, the performance of NCA is impaired by the high rate of false connections in binding information. In this paper, we integrate stability analysis with NCA to form a novel scheme, namely stability-based NCA (sNCA), for regulatory network identification. The method mainly addresses the inconsistency between gene expression data and binding motif information. Small perturbations are introduced to prior regulatory network, and the distance among multiple estimated transcript factor (TF) activities is computed to reflect the stability for each TF's binding network. For target gene identification, multivariate regression and t-statistic are used to calculate the significance for each TF-gene connection. Simulation studies are conducted and the experimental results show that sNCA can achieve an improved and robust performance in TF identification as compared to NCA. The approach for target gene identification is also demonstrated to be suitable for identifying true connections between TFs and their target genes. Furthermore, we have successfully applied sNCA to breast cancer data to uncover the role of TFs in regulating endocrine resistance in breast cancer.

Recent research has revealed various molecular markers in lung cancer. However, the organizational principles underlying their genetic regulatory networks still await investigation. Here we performed Network Component Analysis (NCA) and Pathway Crosstalk Analysis (PCA) to construct a regulatory network in human lung cancer (A549) cells which were treated with 50 uM motexafin gadolinium (MGd), a metal cation-containing chemotherapeutic drug for 4, 12, and 24 hours. We identified a set of key TFs, known target genes for these TFs, and signaling pathways involved in regulatory networks. Our work showed that putative interactions between these TFs (such as ESR1/Sp1, E2F1/Sp1, c-MYC-ESR, Smad3/c-Myc, and NFKB1/RELA), between TFs and their target genes (such as BMP41/Est1, TSC2/Myc, APE1/Sp1/p53, RARA/HOXA1, and SP1/USF2), and between signaling pathways (such as PPAR signaling pathway and Adipocytokines signaling pathway). These results will provide insights into the regulatory mechanism of MGd-treated human lung cancer cells.

A novel pH-responsive poly(amino acid) grafted with oligocation was prepared through the combination of ring-opening polymerization (ROP) and subsequent atom transfer radical polymerization (ATRP). Firstly, poly(γ-2-chloroethyl-L-glutamate) (PCELG) with a pendent 2-chloroethyl group was synthesized through ROP of γ-2-chloroethyl-L-glutamate N-carboxyanhydride (CELG NCA) using n-hexylamine as the initiator. Then, PCELG was used to initiate the ARTP of 2-aminoethyl methacrylate hydrochloride (AMA), yielding poly(L-glutamate)-graft-oligo(2-aminoethyl methacrylate hydrochloride) (PLG-g-OAMA). The pK(a) of PLG-g-OAMA was 7.3 established by the acid-base titration method. The amphiphilic poly(amino acid) could directly self-assemble into a vesicle in PBS. The vesicle was characterized by TEM and DLS. Hydrophilic DOX·HCl was loaded into the hollow core of the vesicle. The in vitro release behavior of DOX·HCl from the vesicle in PBS could be adjusted by the solution pH. In vitro cell experiments revealed that the vesicle could reduce the toxicity of the DOX·HCl. In addition, the preliminary gel retardation assay displayed that PLG-g-OAMA could efficiently bind DNA at a PLG-g-OAMA/DNA weight ratio of 0.3 or above, indicating its potential use as a gene carrier. More in-depth studies of the PLG-g-OAMA vesicle for drug and gene co-delivery in vitro and in vivo are in progress.

Carcinoembryonic antigen (CEA) is still the only routinely used biomarker in colorectal cancer (CRC), but its utility is hampered by poor specificity and sensitivity, and the search for novel biomarkers is highly warranted. The nonspecific cross-reacting antigen 2 (NCA-2), a truncated CEA species molecule which is transcribed from the same gene, has been suggested as an alternative biomarker to CEA. In the present work, specific immunofluorometric assays were used for detection of NCA-2 and full-length CEA in bone marrow plasma from 277 CRC patients to assess their value as prognostic biomarkers, and detection was also performed in tumor tissue and a CRC cell line. Elevated plasma CEA was associated with advanced tumor stage at diagnosis and adverse patient outcome, while for NCA-2, although the same trends were observed, no additional prognostic information was gained. While specific detection of NCA-2 was clearly achieved in plasma samples, cross-reactivity with full-length CEA was observed when the antigen was exposed to common fixation chemicals. The results from this study indicate that NCA-2 is probably not a prognostic biomarker in CRC and, furthermore, underline the issue of antibody specificity when investigating CEA species molecules.

Epithelial carcinoma of the ovary is one of the most common gynecological malignancies and the fifth most frequent cause of cancer death in women. Currently blood test of advanced epithelial tumors are reflected in a high level of CA 125 antigen. However, it is not a good marker for early stage tumors, and may yield false positives. Clearly, there is a need for better understanding of the molecular pathogenesis of epithelial ovarian cancer, so that new drug targets or biomarkers that facilitate early detection can be identified. This work concentrates on finding genetic markers for three epithelial ovarian tumors, using a simple computational method. We give a small set of genetic markers which are able to distinguish clear cell and mucinous ovarian cancers (13 and 26 genes respectively) from other epithelial ovarian tumors with 100% accuracy. We obtain the genes HNF1-beta (TCF2) and GGT1 as the best markers for the clear cell and CEACAM6 (NCA) as the best marker for mucinous ovarian tumors. We employ a feature selection technique based on minimum probability of error for this purpose. We give a ranking of the important genes responsible for these tumors and validate the results using the leave-one-out cross-validation technique. Using this method, we also agree with the common notion that WT1 is one of the best genes to separate serous ovarian tumors from other epithelial ovarian tumors.

BACKGROUND: Many breast, pancreatic, colonic and non-small-cell lung carcinoma lines express CEACAM6 (NCA-90) and CEACAM5 (carcinoembryonic antigen, CEA), and antibodies to both can affect tumor cell growth in vitro and in vivo. Here, we compare both antigens as a function of histological phenotype in breast, pancreatic, lung, ovarian, and prostatic cancers, including patient-matched normal, primary tumor, and metastatic breast and colonic cancer specimens.
METHODS: Antigen expression was determined by immunohistochemistry (IHC) using tissue microarrays with MN-15 and MN-3 antibodies targeting the A1B1- and N-domains of CEACAM6, respectively, and the MN-14 antibody targeting the A3B3 domain of CEACAM5. IHC was performed using avidin-biotin-diaminobenzide staining. The average score +/- SD (0 = negative/8 = highest) for each histotype was recorded.
RESULTS: For all tumors, the amount of CEACAM6 expressed was greater than that of CEACAM5, and reflected tumor histotype. In breast tumors, CEACAM6 was highest in papillary > infiltrating ductal > lobular > phyllodes; in pancreatic tumors, moderately-differentiated > well-differentiated > poorly-differentiated tumors; mucinous ovarian adenocarcinomas had almost 3-fold more CEACAM6 than serous ovarian adenocarcinomas; lung adenocarcinomas > squamous tumors; and liver metastases of colonic carcinoma > primary tumors = lymph nodes metastases > normal intestine. However, CEACAM6 expression was similar in prostate cancer and normal tissues. The amount of CEACAM6 in metastatic colon tumors found in liver was higher than in many primary colon tumors. In contrast, CEACAM6 immunostaining of lymph node metastases from breast, colon, or lung tumors was similar to the primary tumor.
CONCLUSION: CEACAM6 expression is elevated in many solid tumors, but variable as a function of histotype. Based on previous work demonstrating a role for CEACAM6 in tumor cell migration, invasion and adhesion, and formation of distant metastases (Blumenthal et al., Cancer Res 65: 8809-8817, 2005), it may be a promising target for antibody-based therapy.

Lambert-Eaton myasthenic syndrome (LEMS) is a rare autoimmune neuromuscular disorder characterized by pathogenic autoantibodies directed against the presynaptic voltage-gated calcium channels (VGCC), resulting in a clinical syndrome of proximal muscular weakness and autonomic dysfunction. Sixty percent of LEMS cases are associated with cancer, most commonly small cell carcinoma of the lung. In the 40% of LEMS patients without carcinoma, the stimulus for the production of VGCC autoantibodies is unknown; however, these LEMS patients have multiple other organ-specific autoantibodies. To investigate the autoimmune basis of noncancer associated LEMS (NCA-LEMS), high resolution typing of major histocompatibility loci was performed in 23 patients with NCA-LEMS. NCA-LEMS was strongly associated with DRB1*0301 (p<0.0001) and DQB1*0201 (p<0.0001), suggesting that NCA-LEMS is an autoimmune disorder associated with the DR3-DQ2 extended haplotype.

CEA family proteins from human urothelial cell lines of different transformation grades were characterized by flow cytometry and Western blotting using monoclonal antibodies: 26/3/13, D14HD11, 9A6 and 4/3/17. The following observations were made: (i) the urothelial cell lines, representing transformation grade III (TGr III, tumorigenic, invasive cells), were characterized by the presence of a component with molecular mass 110-135 kDa, most probably representing biliary glycoprotein (BGP); (ii) BGP was absent in non-tumorigenic and non-invasive TGr II urothelial cell lines; (iii) a protein band with apparent molecular mass 180 kDa, and migrating as a CEA standard was detected in only one of seven urothelial cell lines analyzed; (iv) a broad band of apparent molecular mass migrating at 65-90 kDa, probably representing NCA-50/90, was found in two tumorigenic and invasive cell lines, HCV 29T and Hu 1703He.

We previously reported a novel monoclonal antibody KOR-SA3544 which predominantly reacted with a surface antigen (sSA3544) expressed on Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL). In the present study, we demonstrate that the antibody specifically recognized nonspecific cross-reacting antigen (NCA)-50/90 (CD66c), one of the carcinoembryonic antigen (CEA)-related glycoproteins encoded by a member of the CEA gene family. In addition, we show that the SA3544 antigen (NCA-50/90) was invariably expressed in cytoplasm of all of the human leukemic cell lines examined (sSA3544-positive B-lymphoid two, sSA3544-negative T or B-lymphoid and non-lymphoid 24) regardless of the presence or absence of surface expression of this antigen. Immunoelectromicroscopic examination revealed that the cytoplasmic antigen was mainly present in granules in sSA3544-positive leukemia cells, whereas it was diffusely present in cytosol in sSA3544-negative leukemia cells. Thus, among members of the CEA family, NCA-50/90 was first demonstrated to be expressed not only on the surface of some leukemia cells, but also in cytoplasm of various types of leukemia cells.

Human non-specific cross-reacting antigen (NCA), a close relative of the tumor marker human carcinoembryonic antigen (CEA), is also an in vitro homotypic intercellular adhesion molecule capable of inhibiting differentiation when expressed ectopically by myoblasts. Moreover, NCA appears to be overexpressed at the transcriptional level to a greater extent and more frequently in colorectal carcinomas than CEA. This study examines the transcriptional control mechanisms responsible for orchestrating NCA expression. The region within 284 bp upstream of the translational start site of the NCA gene was found to be capable of directing high levels of expression in functional promoter assays. Footprinting experiments identified three cis-acting elements and mobility-shift assays revealed that the first of these elements is bound by the upstream stimulating factors USF1 and USF2 while the other two are bound by the stimulatory proteins Sp1 and Sp3. No cis-acting elements corresponding to CEA footprint FP4 or the silencer CEA FP5 were detected in the NCA promoter, which may contribute to the differential expression of NCA versus CEA in tumorigenesis.

IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplification was found for c-erbB-2 in the cell line or in IIB-BR-G(NUDE), although this oncogene was amplified in the patient's primary tumor DNA. The differences observed between the patient's tumor, the cell line and the IIB-BR-G(NUDE) tumors are probably due to clonal expansion of cell variants not present in the original tumor. Electron microscopy of IIB-BR-G growing cells revealed epithelial characteristics with abundant dense granules, presumably secretory, distributed all over the cytoplasm and great nuclear pleomorphism. In vitro, IIB-BR-G cells showed a significant number of invading cells by Matrigel assay. After nearly 40 sequential subcutaneous passages of the original xenograft through nude mice, 80% of recipients developed spontaneous metastases, primarily to the lung and lymph nodes. Since this experimental model allowed to analyze changes produced in cancer cells from the primary tumor during adaptation to in vitro and in vivo growth, our results provide novel insights on the behaviour of hormone independent metastatic breast cancer.

Four human carcinoembryonic antigen (CEA) family members, CEA (CD66e), non-specific cross-reacting antigen (NCA, CD66c), biliary glycoprotein (BGP, CD66a) and CEA gene-family member 2 (CGM2), are expressed in normal mucosal epithelia of the colon. Expression of BGP and CGM2 has recently been demonstrated to be down-regulated in colorectal adenocarcinomas. We have now investigated the expression of the 4 CEA family antigens in gastric adenocarcinoma and carcinoma cell lines in comparison with adjacent normal gastric mucosa. The transcripts of the CEA, NCA and BGP genes evaluated by reverse transcription-polymerase chain reaction were detectable at various levels in all the gastric adenocarcinoma cell lines tested, while CGM2 mRNA was detectable in the cell lines of poorly differentiated but not of well-differentiated carcinomas. The levels of CEA mRNA in normal gastric mucosa were variable but mostly increased in adenocarcinomas. The sparse expression of NCA observed in the normal tissues was markedly up-regulated in the carcinomas. In contrast to previous findings on normal and cancerous colonic tissues, the transcripts of CGM2 were totally undetectable and those of BGP were recognized only marginally, if at all, in normal gastric mucosa, while both messages were detected at significant levels in most of the gastric adenocarcinomas. This was confirmed by in situ hybridization. Our findings indicate that expression of the CEA family antigens, particularly that of BGP and CGM2, is differently regulated in epithelial cells of the colon and the stomach.

Genes coding for CD66a (biliary glycoprotein), carcinoembryonic antigen (CEA) group member 2 (CGM2), and nonspecific cross-reacting antigen (NCA) are members of the human CEA gene subgroup. We investigated a series of 11 colorectal carcinomas by Northern blot and isotopic in situ hybridization (ISH), demonstrating underexpression of CD66a and CGM2 in the majority of the carcinomas as compared with the normal mucosa, whereas NCA was overexpressed. ISH for CD66a and CGM2 mRNA revealed that large areas of the carcinomas remained without or with only faint hybridization signals. However, in every carcinoma, at least some positive foci were observed, indicating remaining cell populations that actively transcribe CD66a and CGM2. In contrast, ISH for NCA displayed strong and extensive autoradiographic signals. By analysis of step sections, foci of CD66a and CGM2 expression were shown to co-localize. Furthermore, these foci contained relatively few nuclei immunohistochemically positive for the proliferation-associated nuclear antigen Ki-67. Our data indicate a dysregulation of the three genes possibly with a common transcriptional control for CD66a and CGM2 and a different control for NCA. The focal expression of CD66a and CGM2 could be interpreted as due to a focal, incomplete, and abortive differentiation or, alternatively, as a consequence of genetic heterogeneity with foci of slow-proliferating subclones.

Among the members of the carcinoembryonic antigen (CEA) family, CD66a (human C-CAM) and CGM2 (CEA gene family member 2) mRNAs are frequently down-regulated in colorectal cancer. In contrast, nonspecific cross-reactive antigen (NCA) mRNA is overexpressed in the majority of these carcinomas. In animal models, the rodent homologues of CD66a have been shown to act as tumor suppressors, suggesting an important role in carcinogenesis. Here we investigate the mRNAs of CD66a, CGM2, and NCA in 22 human colorectal adenomas and the respective normal mucosa specimens by Northern blots. The expression of both CD66a and CGM2 changed in a concomitant fashion. Using oligonucleotides specific for the N-terminal domains, two CD66a transcripts 3.9 and 1.5 kb in size were identified. These showed a greater than 50% down-regulation in 20 of 22 and 18 of 22 adenomas, respectively. Reduction of the CGM2 message was observed in 21 of 22 cases. Complete or near-complete losses of the CD66a 3.9-kb mRNA and the CGM2 message were found in 13 of 22 and 15 of 22 of the tumors, respectively. The medians of CD66a and CGM2 expressions were between 0.3 and 0.0, respectively. The tumor:normal ratio of NCA mRNA expression was increased up to 2.4-fold in 11 of 22 adenomas. Altogether, these results compare well to the changes reported previously for colorectal carcinomas. The high frequency and early appearance of dysregulation of members of the carcinoembryonic antigen family during colorectal tumorigenesis suggests that these changes may be important for the development of the malignant phenotype.

Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLa-CEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.

Carcinoembryonic antigen gene family member 2 (CGM2), a member of the carcinoembryonic antigen (CEA) family, is expressed in normal colon and rectum but is down-regulated in colorectal adenocarcinomas. In situ hybridization studies demonstrate that CGM2 expression is limited to epithelial cells in the upper third of the crypts. Two other CEA family members, biliary glycoprotein (BGP) and nonspecific cross-reacting antigen (NCA), are similarly expressed, whereas CEA transcripts were found down to the base of the crypts but were less predominant in the upper region. Only low CGM2 and BGP mRNA levels were seen in colorectal tumors. CEA mRNA was expressed at an equivalent level in normal epithelia and in tumor cells, whereas NCA transcript levels were upregulated in tumor cells. Monoclonal antibodies that recognize the CGM2 protein reveal its presence on the apical membranes of epithelial cells in the upper third of the crypts but its absence from colorectal tumors, which do express the CEA and NCA-50/90 proteins. The newly cloned CGM2 3'-untranslated region was used to probe RNAs from adenomas, colorectal tumors at different stages of progression, and liver metastases of colorectal adenocarcinomas. This showed that CGM2 is already down-regulated in adenomas when compared to normal mucosae. The CGM2 expression pattern along with its sequence homology to BGP suggests a similar tumor suppressor function for CGM2.

BACKGROUND & AIMS: The nonspecific cross-reacting antigen (NCA) is a cell adhesion molecule, and the messenger RNA for NCA is overexpressed in 92% of colorectal carcinomas. The aim of this study was to determine the cis-acting elements that may be responsible for the expression of NCA.
METHODS: Deletion mutants of the 5' flanking sequence and first intron were ligated into chloramphenicol acetyltransferase expression vectors, transfected into Chinese hamster ovary (CHO), DiFi, and HT-29 human colorectal carcinoma cells, and BxPC-3 and MDAPanc-28 human pancreatic carcinoma cells. The amount of acetylated chloramphenicol was determined to show the presence and activity of cis-acting sequences.
RESULTS: The 5' flanking sequence functions as a promoter in all of cell lines and contains negative regulatory and enhancer sequences. The minimal promoter is active in Chinese hamster ovary and HT-29, though not in MDAPanc-28 cells. The first intron contains a silencer capable of suppressing a heterologous promoter.
CONCLUSIONS: The results show cis-acting sequences within and 5' to the NCA gene, which appear to play a role in the expression of this gene in malignant tissues. Some of these sequences function in a cell type-specific manner. Further studies of these elements may provide insight into the mechanisms of the abnormal growth patterns of malignant cells.

Biliary glycoprotein (BGP) is an adhesion and anti-cell-growth molecule of the carcinoembryonic antigen family. We have earlier demonstrated that BGP mRNA is expressed in hepatocellular carcinomas (HCCs) and the adjacent non-cancerous regions, neither of which express CEA and NCA mRNA. To define an expression level and pattern of BGP at the protein level in HCCs, TS135, a monoclonal antibody (MAb) against BGP, was prepared. This MAb clearly reacted with BGP with a molecular weight of 110 kDa and 85 kDa (BGP-110/85). It cross-reacted weakly with NCA-90 from NCA transfectants, but not at all with CEA-200 from the serum of a colon-cancer patient. The BGP transfectants of cultured hepatocellular carcinoma cHc-4 cells showed Ca2+-dependent cell aggregation, which was partially inhibited by modulating BGP on the cell surface with MAb TS135. Immunostaining of non-cancerous liver tissues with MAb TS135 indicated that BGP could be expressed in the bile canalicular domain of hepatocytes. In HCCs, the expression of BGP was predominantly found in the well-differentiated type, where the bile canaliculi and the apical portion of pseudoglands were positively stained, although their staining intensity and stained area were lower and more limited, respectively, than those of non-cancerous regions. The percentage of faintly positive and negative cases (n = 22) from the total (n = 30) was 73%. This suggests that the expression level of BGP decreased in HCCs as compared with adjacent non-cancerous regions.

The heterophilic cell adhesion mediated by CD66b (carcinoembryonic antigen (CEA) gene family member 6, CGM6) and CD66c (nonspecific cross-reacting antigen, NCA), both CEA family members expressed on neutrophils, was investigated using their soluble recombinant proteins prepared in silkworm larvae. The recombinant CD66b and CD66c immobilized on plastic bound CHO transfectants expressing CD66c and CD66b, respectively. Their deglycosylated forms retained the adhesion activity, suggesting that their carbohydrate portions are not prerequisite for the binding. This cell adhesion appeared to be mediated via interaction between the N domains of CD66b and CD66c, because CD66 antibodies recognizing their N domains inhibited the binding. Neutrophils, when activated, adhered to the immobilized CD66b and CD66c. In addition, the binding of primed neutrophils to the antigens induced superoxide anion release. The cell adhesion mediated by CD66b and CD66c may play a role in interaction between neutrophils or between neutrophils and epithelial cells expressing CD66c in vivo.

The carcinoembryonic antigen (CEA) and the classical non-specific cross-reacting antigens (NCAs) belong to the CEA gene family which is part of the immunoglobulin superfamily. In normal hematopoiesis, CEA gene family members (CGMs) have only been reported on cells of myeloid and monocytic origin. In the present study, we analyzed 62 childhood acute lymphoblastic leukemias (ALLs) and seven surface immunoglobulin positive (sig+) B-cell lines for the expression of the CEA family members CEA, NCA-50/90, NCA-95, NCA-160, CGM1 and CGM7. We demonstrated that members of the CEA family were present in 76% of childhood ALLs of B- and T-cell origin. In ALLs of B-cell origin, 82% of the samples expressed at least one CEA subgroup member: 38% NCA-50/90 (CD66c), 31% NCA-160 (CD66a), and 13% both. Six of seven B-cell lines solely expressed NCA-160. In seven ALL of T-cell origin, sole NCA-160 expression was present in 29% of the cases. CEA and CGM1 were not expressed in childhood ALLs or in the sIg+ B-cell lines. In 15 ALLs and seven B-cell lines which could be analyzed for CGM7 expression, the antigen was not detected. NCA-95 was not expressed in 91% of the B-lineage ALLs, in T-lineage ALLs and in the B-cell lines. However, five B-lineage ALLs showed conflicting data on the binding patterns of two, on leukocytes specifically NCA-95 recognizing antibodies suggesting either expression of unknown forms of NCA-95 or NCA-50/90 or of a yet unknown member of the CEA family in these ALL cells. The expression of CEA subgroup members in childhood ALL cells might have prognostic impacts, as an inverse correlation exists between NCA expression on leukemic blasts and the risk factor white blood count at diagnosis.