Charng-Jui Chen

CEACAM1-4S, a cell–cell adhesion molecule, mediates apoptosis and reverts mammary carcinoma cells to a normal morphogenic phenotype in a 3D culture

In a 3D model of breast morphogenesis, CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) plays an essential role in lumen formation in a subline of the nonmalignant human breast cell line (MCF10A). We show that mammary carcinoma cells (MCF7), which do not express CEACAM1 or form lumena when grown in Matrigel, are restored to a normal morphogenic program when transfected with CEACAM1-4S, the short cytoplasmic isoform of CEACAM1 that predominates in breast epithelia. During the time course of lumen formation, CEACAM1-4S was found initially between the cells, and in mature acini, it was found exclusively in an apical location, identical to its expression pattern in normal breast. Lumena were formed by apoptosis as opposed to necrosis of the central cells within the alveolar structures, and apoptotic cells within the lumena expressed CEACAM1-4S. Dying cells exhibited classical hallmarks of apoptosis, including nuclear condensation, membrane blebbing, caspase activation, and DNA laddering. Apoptosis was mediated by Bax translocation to the mitochondria and release of cytochrome c into the cytoplasm, and was partially inhibited by culturing cells with caspase inhibitors. The dynamic changes in CEACAM1 expression during morphogenesis, together with studies implicating extracellular matrix and integrin signaling, suggest that a morphogenic program integrates cell–cell and cell–extracellular matrix signaling to produce the lumena in mammary glands. This report reveals a function of CEACAM1-4S relevant to cellular physiology that distinguishes it from its related long cytoplasmic domain isoform.

Role of interferon regulatory factor-1 in the induction of biliary glycoprotein (cell CAM-1) by interferon-gamma.

Biliary glycoprotein (BGP), also known as C-CAM-1, has been shown to be down-regulated in colon and prostate tumors. Previously, we demonstrated that BGP mRNA is up-regulated by interferon-γ (IFN-γ) in colon cancer cell lines (Takahashi, H., Okai, Y., Paxton, R. J., Hefta, L. J. F., and Shively, J. E. (1993) Cancer Res. 53, 1612-1619). We now show that the BGP promoter contains an interferon-sensitive response element (ISRE) that is specifically protected in in vivo footprints. Interferon regulatory factor-1 (IRF-1) was identified as the ISRE-binding factor by electrophoretic mobility shift assays. The induction of IRF-1 mRNA by IFN-γ in HT-29 cells reaches a maximum at 6 h and is superinduced by cycloheximide. Four mRNA species for BGP are induced by IFN-γ, the major band of which is inhibited by cycloheximide. Transfection of HT-29 cells with an IRF-1 expression plasmid (pAct-1) transactivates a BGP promoter reporter gene containing wild-type (but not mutant) ISRE. Electrophoretic mobility shift assay analysis of a second footprint reveals the binding of Sp1, an Sp1-like protein, and upstream stimulatory factor. The Sp1-like complex was also induced by IFN-γ treatment of HT-29 cells and may be a second point of transcriptional control for the BGP gene.

Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe454) and lysine (Lys456) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe454 at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr457 and/or Ser459 are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling.

Direct Interaction of Tumor Suppressor CEACAM1 with Beta Catenin: Identification of Key Residues in the Long Cytoplasmic Domain

CEACAM1-4L (carcinoembryonic antigen cell adhesion molecule 1, with 4 extracellular Ig-like domains and a long, 71 amino acid cytoplasmic domain) is expressed in epithelial cells and activated T-cells, but is down-regulated in most epithelial cell cancers and T-cell leukemias. A highly conserved sequence within the cytoplasmic domain has ca 50% sequence homology with Tcf-3 and −4, transcription factors that bind β-catenin, and to a lesser extent (32% homology), with E-cadherin that also binds β-catenin. We show by quantitative yeast two-hybrid, BIAcore, GST-pull down, and confocal analyses that this domain directly interacts with β-catenin, and that H-469 and K-470 are key residues that interact with the armadillo repeats of β-catenin. Jurkat cells transfected with CEACAM1-4L have 2-fold less activity in the TOPFLASH reporter assay, and in MCF7 breast cancer cells that fail to express CEACAM1, transfection with CEACAM1 and growth in Ca2+ media causes redistribution of β-catenin from the cytoplasm to the cell membrane, demonstrating a functional role for the long cytoplasmic domain of CEACAM1 in regulation of β-catenin activity.

Role of calpain-9 and PKC-δ in the apoptotic mechanism of lumen formation in CEACAM1 transfected breast epithelial cells

CEACAM1-4S (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I membrane protein with a short (12-amino acid) cytoplasmic tail. Wild type CEACAM1-4S-transfected MCF7 cells form glands with lumena when grown in 3D culture, while null mutations of two putative phosphorylation sites (T457A and S459A) in the cytoplasmic domain fail to undergo lumen formation. When gene chip analysis was performed on mRNA isolated from both wild type and T457A,S459A mutated CEACAM1-4S-transfected MCF7 cells grown in 3D culture, calpain-9 (CAPN9) was identified out of over 400 genes with a >2 log 2 difference as a potential inducer of lumen formation. Inhibition of CAPN9 expression in MCF7/CEACAM1-4S cells by RNAi or by calpeptin or PD150606 inhibited lumen formation. Transfection of CAPN9 into wild type MCF7 cells restores lumen formation demonstrating that calpain-9 may play a critical role in lumen formation. Additionally, we demonstrate that the apoptosis related kinase, PKC-delta, is activated by proteolytic cleavage during lumen formation exclusively in wild type CEACAM1-4S-transfected MCF7 cells grown in 3D culture and that lumen formation is inhibited by either RNAi to PKC-delta or by the PKC-delta inhibitor rottlerin.

Regulation of CEACAM1 transcription in human breast epithelial cells

BackgroundCarcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast).ResultsUsing in vivo footprinting and chromatin immunoprecipitation experiments we show that the CEACAM1 proximal promoter in breast cells is bound in its active state by SP1, USF1/USF2, and IRF1/2. When down-regulated the CEACAM1 promoter remains accessible to USF2 and partially accessible to USF1. Interferon-γ up-regulates CEACAM1 mRNA by a mechanism involving further induction of IRF-1 and USF1 binding at the promoter. As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells. The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure.ConclusionsOur data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.

Mutational analysis of the cytoplasmic domain of CEACAM1-4L in humanized mammary glands reveals key residues involved in lumen formation: Stimulation by Thr-457 and inhibition by Ser-461

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion, undergoes extensive alternative splicing, resulting in isoforms with 1-4 Ig-like extracellular domains (ECDs) with either long or short cytoplasmic tails. We have previously shown that CEACAM1-4L (4 ECDs with a long cytoplasmic domain) formed glands with lumena in humanized mammary mouse fat pads in NOD/SCID mice. In order to identify the key residues of CEACAM1-4L that play essential roles in lumen formation, we introduced phosphorylation mimic (e.g., Thr-457 or Ser-461 to Asp) or null mutations (Thr-457 or Ser-461 to Ala) into the cytoplasmic domain of CEACAM1-4L and tested them in both the in vivo mouse model and in vitro Matrigel model of mammary morphogenesis. MCF7 cells stably expressing CEACAM1-4L with the single mutation T457D or the double mutant T457D+S461D, but not the null mutants induced central lumen formation in 3D Matrigel and in humanized mammary fat pads. However, the single phosphorylation mimic mutation S461D, but not the null mutation blocked lumen formation in both models, suggesting that S461 has inhibitory function in glandular lumen formation. Compared to our results for the -4S isoform (Chen et al., J. Biol. Chem, 282: 5749-5760, 2008), the T457A null mutation blocks lumen formation for the -4L but not for the -4S isoform. This difference is likely due to the fact that phosphorylation of S459 (absent in the -4L isoform) positively compensates for loss of T457 in the -4S isoform, while S461 (absent in the -4S isoform) negatively regulates lumen formation in the -4L isoform. Thus, phosphorylation of these key residues may exert a fine control over the role of the -4L isoform (compared to the -4S isoform) in lumen formation.

The adaptor SASH1 acts through NOTCH1 and its inhibitor DLK1 in a 3D model of lumenogenesis involving CEACAM1

ABSTRACT CEACAM1 transfection into breast cancer cells restores lumen formation in a 3D culture model. Among the top up‐regulated genes that were associated with restoration of lumen formation, the adaptor protein SASH1 was identified. Furthermore, SASH1 was shown to be critical for lumen formation by RNAi inhibition. Upon analyzing the gene array from CEACAM1/MCF7 cells treated with SASH1 RNAi, DLK1, an inhibitor of NOTCH1 signaling, was found to be down‐regulated to the same extent as SASH1. Subsequent treatment of CEACAM1/MCF7 cells with RNAi to DLK1 also inhibited lumen formation, supporting its association with SASH1. In agreement with the role of DLK1 as a NOTCH1 inhibitor, NOTCH1, as well as its regulated genes HES1 and HEY1, were down‐regulated in CEACAM1/MCF7 cells by the action of DLK1 RNAi, and up‐regulated by SASH1 RNAi. When CEACAM1/MCF7 cells were treated with a &ggr;‐secretase inhibitor known to inhibit NOTCH signaling, lumen formation was inhibited. We conclude that restoration of lumen formation by CEACAM1 regulates the NOTCH1 signaling pathway via the adaptor protein SASH1 and the NOTCH1 inhibitor DLK1. These data suggest that the putative involvement of NOTCH1 as a tumor‐promoting gene in breast cancer may depend on its lack of regulation in cancer, whereas its involvement in normal lumen formation requires activation of its expression, and subsequently, inhibition of its signaling.

ETS transcription factor ELF5 induces lumen formation in a 3D model of mammary morphogenesis and its expression is inhibited by Jak2 inhibitor TG101348

&NA; The loss of expression of a single gene can revert normal tissue to a malignant phenotype. For example, while normal breast has high lumenal expression of CEACAM1, the majority of breast cancers exhibit the early loss of this gene with the concurrent loss of their lumenal phenotype. MCF7 cells that lack CEACAM1 expression and fail to form lumena in 3D culture, regain the normal phenotype when transfected with CEACAM1. In order to probe the mechanism of this gain of function, we treated these cells with the clinically relevant Jak2 inhibitor TG101348 (TG), expecting that disruption of the prolactin receptor signaling pathway would interfere with the positive effects of transfection of MCF7 cells with CEACAM1. Indeed, lumen formation was inhibited, resulting in the down regulation of a set of genes, likely involved in the complex process of lumen formation. As expected, inhibition of the expression of many of these genes also inhibited lumen formation, confirming their involvement in a single pathway. Among the genes identified by the inhibition assay, ETS transcription factor ELF5 stood out, since it has been identified as a master regulator of mammary morphogenesis, and is associated with prolactin receptor signaling. When ELF5 was transfected into the parental MCF7 cells that lack CEACAM1, lumen formation was restored, indicating that ELF5 can replace CEACAM1 in this model system of lumenogenesis. We conclude that the event(s) that led to the loss of expression of CEACAM1 is epistatic in that multiple genes associated with a critical pathway were affected, but that restoration of the normal phenotype can be achieved with reactivation of certain genes at various nodal points in tissue morphogenesis.