CEACAM1 long isoform has opposite effects on the growth of human mastocytosis and medullary thyroid carcinoma cells

Abstract

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in a number of tumor cell types. The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing isoforms of this molecule which possess a long cytoplasmic tail (CEACAM1-L) generally play inhibitory roles in cell function by interacting with Src homology 2 domain-containing tyrosine phosphatase (SHP)-1 and/or SHP-2. Src family kinases (SFKs) are also known to bind to and phosphorylate CEACAM1-L isoforms. Here, we report that CEACAM1 was uniquely expressed at high levels in both human neoplastic mast cells (mastocytosis) and medullary thyroid carcinoma cell (MTC) lines, when compared with their expression in nonneoplastic mast cells or nonneoplastic C cells. This expression was mainly derived from CEACAM1-L isoforms based upon assessment of CEACAM1 mRNA expression. CEACAM1 knockdown upregulated cell growth of HMC1.2 cells harboring KIT mutations detected in clinical mastocytosis, whereas downregulated the growth of TT cells harboring RET mutations detected in clinical MTCs. Immunoblotting, ELISA and immunoprecipitaion analysis showed that activated SHP-1 is preferentially associated with CEACAM1 in HMC1.2 cells harboring KIT mutations, whereas Src family kinases (SFKs) are preferentially associated with CEACAM1 in TT cells harboring RET mutations. These studies suggest that the dominantly interacting proteins SHP1 or SFK determine whether CEACAM1-L displays a positive or negative role in tumor cells.

Keywords: CEACAM1; SHP-1; Src family kinases; mast cell; medullary thyroid carcinoma.

Figure 1

Human neoplastic mast cells express CEACAM1. (A) Primer design for reverse transcription PCR. (B) Reverse transcription PCR (representative bands) and real‐time PCR (relative copy numbers). (C) Western blotting for the neoplastic mast cell lines. The K562 cells were used as a positive control and the Jurkat cells as a negative control. (D) Flow cytometry for HMC1.2 and HuMCs. (E) Reverse transcription PCR (representative bands) and real‐time PCR (relative copy numbers). (F) Western blotting for HuMCs. Arrowheads indicate the expected size of CEACAM1 protein. (G) Immunostaining of skin mastocytosis cells derived from a patient with mastocytosis or chronic dermatitis, nonspecific. Both mastocytosis cells and nonneoplastic mast cells were shown as tryptase‐positive cells (red arrowheads), and the corresponding cells are also indicated by the red arrowheads in the right column. Bars: 50 μm.

Figure 2

Human medullary thyroid carcinoma cells express CEACAM1. Human medullary thyroid carcinoma cell line TT expresses CEACAM1. (A) Reverse transcription PCR (representative bands) and real‐time PCR (relative copy numbers), (B) Western blotting, and (C) Immunocytochemistry were performed. The K562 cells were used as a positive control and the Jurkat cells as a negative control. (D) Immunostaining of clinical medullary thyroid carcinoma cells (Case 1 & 2) and nonneoplastic C cells (red arrowheads, Case 3). Bars: 50 μm.

Figure 3

CEACAM1 knockdown enhances cell growth of LAD2, HMC1.1 and HMC1.2 cells, but suppresses that of TT cells. (A) Establishment of CEACAM1‐knockdown LAD2, HMC1.1, and HMC1.2 and TT cells. Reverse transcription PCR, real‐time PCR (relative copy numbers), Western blotting, and morphology (LAD2, HMC1.1 and HMC1.2 cells; Papanicolaou staining, TT cells; Diff‐Quik stain). (B) The CCK‐8 kit was used for the evaluation of the cell growth of CEACAM1‐knockdown or mock LAD2, HMC1.1, HMC1.2 and TT for the indicate time (n = 3, respectively). *P < 0.05, when compared with the value of mock cells.

Figure 4

Activated SHP‐1 is preferentially associated with CEACAM1 in HMC1.2 cells, whereas activated SFKs are preferentially associated with CEACAM1 in TT cells. The phosphorylation status of SFKs and SHP‐1 in CEACAM1‐knockdown and mock HMC1.2 or TT cells. Western blotting. Data are representative of three individual experiments. (B) ELISA for phospho‐SHP‐1 or phospho‐SFKs in HMC1.2 or TT cells. Relative values when the values of mock cells are 100. *P < 0.05, when compared with the value of mock cells. (C) The interaction of SFKs or SHP‐1 with CEACAM1 in HMC1.2 and TT cells. Left panel: Immunoprecipitation. Right panel: the average of relative values of the band intensities in the blots (n = 3), when the band intensities of HMC1.2 are normalized to 100. (D) The CCK‐8 kit was used for the evaluation of the effect of the specific inhibitor for SFKs PP1 and the specific inhibitor for SHP‐1 PTP inhibitor I on the cell growth of HMC1.2 (24 h) and TT (48 h) (n = 3, respectively). *P < 0.05, when compared with the value of mock cells.