CEACAM1 activation with an agonistic monoclonal antibody inhibits colon cancer cell growth via the L isoform.

Introduction

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an inhibitory receptor expressed on structural and immune cells and on various tumor cells. CEACAM1 is activated by heterologous or homogenous CEACAM1-CEACAM1 interactions. By their CEACAM1, tumor cells bind and activate CEACAM1 expressed on immune cells, thereby inhibiting potential anti-tumor immunity. CEACAM1 has two isoforms - CEACAM1-L (long) and CEACAM1-S (short) cytoplasmic domains. The L isoform possesses immune receptor tyrosine-based inhibitory motifs (ITIMs) and therefore exerts an inhibitory role, while the function of S isoform is yet unclear. In our previous study, we developed CCM5.01, an activating anti-CEACAM1 mAb, that inhibited melanoma cell growth and induced cell death. In this study, we aimed to test whether CCM5.01 could inhibit colon cancer cell lines as a function of their CEACAM1 L and S expression.

Materials and methods

After confirming the expression of CEACAM1 on the parental HT-29 colon cancer cells and observing a decrease in cell viability following activation with CCM5.01, HT-29 cells were transfected with either h-CEACAM1-4L (HT-4L) or h-CEACAM1-4S (HT-4S) to get strong, stable and uniform expression. Transfected cells were treated with CCM5.01 (72hrs) and cell viability was assessed by MTT assay and Trypan Blue or propidium Iodide (PI, FACS analysis). In addition, IL-8 and IL-33 release was analyzed by ELISA and CEACAM1 signal transduction by WB. Finally, transfected cells were seeded within spheroid molds to examine the effect of CCM5.01 in a 3D model.

Results and discussion

Our results show a significant decrease in the viability of HT-4L cells accompanied by reduced IL-8 and IL-33 release following treatment with CCM5.01. Additionally, CCM5.01 treatment induced SHP1 and p53 phosphorylation and caspase-3 activation leading to cell apoptosis. The spheroid 3D model also demonstrated reduced viability of HT-4L cells. In contrast, CCM5.01 treatment of HT-4S cells resulted in increased cell viability, cytokine release, and larger 3D spheroids implying cell activation.

Conclusions
Activating CEACAM1 in HT-29 colon cancer cells reduced cell growth. Isoform-unique analysis revealed that the inhibitory form is CEACAM1-L while CEACAM1-S is likely activating, therefore we suggest that CEACAM1-L isoform is a potential therapeutic target in colon cancer.