Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation

CEACAM1-3S Drives Melanoma Cells into NK Cell-Mediated Cytolysis and Enhances Patient Survival

Abstract CEACAM1 is a widely expressed multifunctional cell–cell adhesion protein reported to serve as a poor prognosis marker in melanoma patients. In this study, we examine the functional and clinical contributions of the four splice isoforms of CEACAM1. Specifically, we present in vitro and in vivo evidence that they affect melanoma progression and immune surveillance in a negative or positive manner that is isoform specific in action. In contrast with isoforms CEACAM1-4S and CEACAM1-4L, expression of isoforms CEACAM1-3S and CEACAM1-3L is induced during disease progression shown to correlate with clinical stage. Unexpectedly, overall survival was prolonged in patients with advanced melanomas expressing CEACAM1-3S. The favorable effects of CEACAM1-3S related to enhanced immunogenicity, which was mediated by cell surface upregulation of NKG2D receptor ligands, thereby sensitizing melanoma cells to lysis by natural killer cells. Conversely, CEACAM1-4L downregulated cell surface levels of the NKG2D ligands MICA and ULBP2 by enhanced shedding, thereby promoting malignant character. Overall, our results define the splice isoform-specific immunomodulatory and cell biologic functions of CEACAM1 in melanoma pathogenesis. Cancer Res; 75(9); 1897–907. ©2015 AACR.</jats:p

Characterization of the CEACAM1 isoform pattern as determined by quantitative RT-PCR of the two A549 subpopulations.

<p>Characterization of the CEACAM1 isoform pattern as determined by quantitative RT-PCR of the two A549 subpopulations.</p

CEACAM1-L but not CEACAM1-S secures cell contact-inhibition.

<p>A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a> using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.</p

CEACAM6 acts as an inducer of cellular proliferation in confluent A549.

<p>A) The expression of the proliferation marker Ki67 is limited to A549-T cells that also expressed CEACAM6 on their cell surface. Confluent CEACAM6-negative and CEACAM6-positive A549-T cells were separated by mAb 9A6 loaded magnetic protein G microbeads and μMAS magnetic sorting columns (Miltenyi Biotec) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. Immunoblot analysis of confluent CEACAM6-negative and CEACAM6-positive A549-T cell lysates was performed applying a Ki67 specific antibody followed by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The data shown are representative of three separate experiments. B) Cell cycle analysis of CEACAM6 negative and CEACAM6 positive A549-T cells. CEACAM6-negative and CEACAM6-positive A549 cells were fixed in 80% ethanol and stained with propidium iodide, and analyzed by flow cytometry as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>.” The DNA content in the different cell fractions is given in arbitrary units on the X-axis. Cells in the G2-M phase (second peak) contained twice as much DNA as cells in the G0-G1 phase (first peak). Cells between the peaks represent cells in the S-phase. The relative proportions of cells in the various phases are shown above the DNA profiles. Filled curve, CEACAM6 negative A549 cells; thick curve, CEACAM6 positive A549 cells. C) Confluent control sh-plasmid transfected A549-T (A549-shControl) and shCEACAM6 transfected A549-T cells (A549-shCC6) were stained for CEACAM1 with mAb clone 283340 and for CEACAM6 with mAb 9A6 (thick lines). The background fluorescence was determined by incubating the cells with control IgG antibody (thin lines). Samples were analyzed by flow cytometry. Compared to A549-shControl cells, A549-shCC6 cells completely lacked CEACAM6 expression, but continued to express CEACAM1. D) Phase contrast images of (a) control sh-plasmid transfected A549-T and (b) shCEACAM6 transfected A549-T cells. Confluent control sh-plasmid transfected A549-T piled up and formed unanchored spheroidal cell aggregates on top of the monolayer revealing insufficient contact inhibition. (b) In contrast, A549-shCC6 cells formed well spread monolayers without detection of unanchored, spheroidal cell growth indicative tight contact inhibition. Bar, 50 µm.</p

Representative images of different A549 cell clones demonstrating their morphological heterogeneity by phase contrast imaging.

<p>Subpopulation A549-NT grown exponentially (a), to tight confluence forming cell-contact inhibited monolayers (b). Tight confluent subpopulation of A549-T (c) piled up on the monolayer revealing the ability to survive and proliferate independently of cell-matrix anchorage, leading to detached spheroidal colonies on top of the monolayer. (d) Spheroidal A549-T colonies seeded into fresh culture vessels attached and grew as monolayers. Bar, 50 µm.</p

Characterization of the cell surface expression of different CEACAMs in A549-NT and A549-T cells.

<p>A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a> section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.</p